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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 I T S SUBSEQUENT USE BY ACETOBACTER SUBOXYDANS.  A Thesis submitted i n P a r t i a l F u l f i l l m e n t o f t h e R e q u i r e m e n t s f o r t h e Degree o f MASTER OF SCIENCE IN AGRICULTURE in the Department o f A n i m a l Husbandry.  The  U n i v e r s i t y of B r i t i s h Columbia August, 1949.  INFLUENCE OF ULTRAVIOLET IRRADIATION OF n-AMINOBENZOIC ACID AND I T S SUBSEQUENT USE BY ACETOBACTER SUBOXYDANS.  Warren D . K i t t s  ABSTRACT T h i s s t u d y h a s b e e n b a s e d 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 the 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 is  attributable  violet It  t o an a b s o r p t i o n o f e n e r g y i n t h e  r e g i o n b y one o r more e s s e n t i a l c e l l u l a r  i s further  molecular  suggested t h a t the  s h i f t i n the  absorbing metabolite  structurally s t i l l  different  w i t h the normal m e t a b o l i c f u n c t i o n s o f the 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 literature  ultraviolet  light  and t h e  for  altered its  to  interfere  cell*  Facteri-  ensues*  study of p-aminobenzoic a c i d  evidence to  related  subjects  establishment  and  gives  s u p p o r t t h e above h y p o t h e s i s  been r e v i e w e d and i s p r e s e n t e d b y s e c t i o n s The  case  a s s o c i a t e d w i t h the b i o l o g i c a l a c t i o n of  sulphonamides as w e l l as o t h e r circumstantial  a  (in this  quite s i m i l a r to  metabolic precursor, i s s u f f i c i e n t l y  The  metabolites.  absorption produces  p-aminobenzoic a c i d ) o f such a n a t u r e t h a t the compound, w h i l e  ultra-  i n the  of a m i c r o b i o l o g i c a l assay  has  thesis* procedure  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 was done i n  order 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 able to  produce  an a l t e r a t i o n  i n the  p-aminobenzoic acid molecule.  A m i c r o b i o l o g i c a l assay procedure  for the  deter-  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 h a s b e e n recommended, was f o u n d n e c e s s a r y cedures.  It  be f o l l o w e d mentations I n the  to modify the  was s u g g e s t e d i n future  reported  published assay  l i n e s of the  s t u d y o f the  upon w h i c h the  p r e s e n t work i s based i s has  of p-aminobenzoic acid no l o n g e r a c t suboxydans.  to  conclude that  been d e m o n s t r a t e d so a l t e r s  the  and t h e  it  can  Acetobacter  It  effect  Acetobacter i f  the  was a b l e  chemical action of u l t r a v i o l e t  a c i d were c a r r i e d out w i t h t h e  of i r r a d i a t e d  irradiation  show t h e  observe  to  light  Determination of  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  Spectrophotometer.  least,  irradiations.  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 . absorption  for  p-aminobenzoic a c i d ,  a c t i o n s of these  A study o f the  cells of  subsequent study to  "essential metabolite", the  that  compound t h a t  c a r r i e d out to  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  counteract  hypothesis  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  suboxvdans.  the  sound, at  as an " e s s e n t i a l m e t a b o l i t e "  under  experi-  biological action of ultraviolet  seems s a f e  i n that i t  should  herein.  irradiation i t  in part,  it  pro-  t h a t t h i s recommendation  work a l o n g t h e  as  a i d o f the  was shown t h a t t h e  the  p-aminobenzoic Beckman M o d e l DU absorption  curve  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  that of normal p-aminobenzoic  acid*  • K P r e l i m i n a r y attempts to i s o l a t e the 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 the out and r e c o r d e d . the  i d e n t i t y o f the  acid;  however f u t u r e  successful pure  It  w o r k w h i c h i s p l a n n e d may be compound o r compounds i n a  result  the  i d e n t i t y o f these  established*  The o r i g i n a l h y p o t h e s i s e m p h a s i z e d t h e t h a t the  a c i d would act  l i k e l y that  emphasized  an a n t i - m e t a b o l i t e  "essential metabolite",  The work  reported, It  does  i s f o r m e d o r more p-aminobenzoic  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 biological  the  of p-aminobenzoic  such a c o n t e n t i o n i n e n t i r e t y .  either the  i r r a d i a t i o n product  as. a n a n t i - m e t a b o l i t e .  does not support suggest that  state  i r r a d i a t i o n product of p-aminobenzoic  s t a t e and w i t h t h i s  likelihood  carried  i s i m p o s s i b l e at present to  i n i s o l a t i n g the  compounds may be  new compound were  assay.  i  acid,  ACKNOWLEDGEMENT  The w r i t e r wishes t o take t h i s  opportunity  to thank P r o f e s s o r H.M. King, Head o f . t h e of Animal Husbandry  Department  f o r p r o v i d i n g the f a c i l i t i e s  i n order t h a t t h i s work c o u l d be accomplished. S i n c e r e thanks a r e expressed t o Dr. A . J . Wood, A s s o c i a t e P r o f e s s o r i n the Department of Animal Husbandry,  f o r suggesting t h i s  and a l s o f o r h i s d i r e c t i o n ,  problem  a s s i s t a n c e and c r i t i c i s m  during t h e course of t h i s study. The w r i t e r a l s o wishes t o express h i s g r a t i t u d e t o t h e Defence Research Board of Canada f o r t h e i r f i n a n c i a l a s s i s t a n c e , and t o the B r i t i s h Columbia Research C o u n c i l f o r the use of c e r t a i n equipment work.  essential  t o t h e conductance of the  TABLE OF CONTENTS  I.  Introduction  1  I I . L i t e r a t u r e Review: A. A c t i o n of 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 B. A Hypothesis Proposed t o E x p l a i n the E f f e c t s of U l t r a v i o l e t Energy on C e l l u l a r Metabolism C. Enlargement of the Working Hypothesis to 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 Energy on C e l l u l a r . Metabolism D. p-Aminobenzoic A c i d ; Mode of A c t i o n of ' Sulphonamides E. R e l a t i o n Between Chemical 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 of a Compound F. Other 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 . . G. p-Aminobenzoic A c i d and i t s Relat ion to Pteroylglutamic Acid i n B a c t e r i a l Nutrition. H. Review o f the R e l a t i o n s h i p Between pAminobenzoic Acid and Sunburn I. P r o t e c t i o n from Sunburn by p-Aminobenzoic Acid  3 7 10 14 19 22 25 30 39  I I I . Experimental A. I n t r o d u c t i o n B. Establishment of the Assay Procedure C. B i o l o g i c a l Aspects i n R e l a t i o n t o Assay . Procedure: Growth Requirements for A. suboxydans. . D. C o r r e l a t i o n of T u r b i d i t y Readings with A c t u a l C e l l Count E. F i n a l Re"commended Assay Procedure F. 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 Irradiation G. The Chemical A c t i o n o f U l t r a v i o l e t Light on p-Aminobenzoic A c i d H. P r e l i m i n a r y Tests t o I s o l a t e the I r r a d i a t i o n Product or Products of pAminobenzoic A c i d IV. Summary  40 41 56 64 68 68 86 94 95  Table of Contents continued;  V. Appendices:  Page  A. Appendix I : B. Appendix  II:  C. Appendix I I I : D. Appendix  IV:  E. Appendix V: F. Appendix VI:  .VI.  Bibliography  Review of Photochemical Reaction The Use of Microorganisms t o Assay Vitamins Procedures f o r M i c r o b i o l o g i c a l and Chemical Assay, of p-Aminobenzoic A c i d Composition of Media Used i n the Present Work Determination of the Number of B a c t e r i a l C e l l s i n the I n oculum . D e s c r i p t i o n and Use of the General E l e c t r i c , Mercury A r c , 15 watt G e r m i c i d a l Lamp  98 103 113 119 120 122 124  INTRODUCTION . I t has  been recognized  f o r the  past  h a l f century  u l t r a v i o l e t l i g h t i s capable of d e s t r o y i n g molds (Heyroth, 1941).  The  bacteria  that  and  many s t u d i e s on the mechanism  of the l e t h 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 which have been  c a r r i e d out,  an adequate  have f a i l e d to provide  f o r the b i o l o g i c a l a c t i o n of these r a y s .  explanation  Extensive  reviews  have been w r i t t e n around t h i s general- s u b j e c t . • (Blum, Heyroth, 1941;  S a l l e , 1943.)  It i s of i n t e r e s t to note the work of H a r r i s  and  Hoyt (1919) i n r e l a t i o n to the a c t i o n of u l t r a v i o l e t on b a c t e r i a .  These workers came to the c o n c l u s i o n  aromatic amino acids,- tryptophan-e, -t-ryo-si-ne and line,  present  i n the b a c t e r i a l c e l l s ,  those l i g h t rays  are able  that are l e t h a l i n a c t i o n .  metabolic  b r i n g about an i n h i b i t i o n of the  that.the  to absorb absorption  or may  cell's  years attempts to  the b a c t e r i o s t a t i c a c t i o n of i n i m i c a l agents on the of an i n t e r f e r e n c e by these agents with c e r t a i n metabolites."  The  now  c l a s s i c a l theory  a c t i o n proposed by F i l d e s (1940) and  cept.  in  functions.  A concept proposed i n recent  v i d e s an  light  phenyla-  The  of such rays re.sults i n the death of the c e l l , some cases,  1941;  explain basis  "essential  of sulphonamide  by Woods (1940) pro-  e x c e l l e n t example of the a p p l i c a t i o n of t h i s  con-  - 2 -  The  present work i s based  which proposes light  on a working hypothesis  t h a t the b a c t e r i c i d a l a c t i o n of  ultraviolet  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 the  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 metabolites.  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 pro-  duces a molecular s h i f t i n the absorbing m e t a b o l i t e of such a nature that the a l t e r e d compound while still  structurally  q u i t e s i m i l a r t o i t s metabolic 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 the normal metabolic f u n c t i o n s of  the c e l l .  death may If  B a c t e r i o s t a s i s then f o l l o w s and  ultimately  result. such a theory proves t e n a b l e then c e r t a i n p o i n t s  must be e s t a b l i s h e d from p u b l i s h e d work and by new e x p e r i mental p r o o f .  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 the  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  bio-  and other r e l a t e d sub-  j e c t s has been surveyed and i s given by s e c t i o n s i n a review f o l l o w i n g t h i s that u l t r a v i o l e t  Introduction.  l i g h t has been u t i l i z e d  and commercial purposes and Food Technology.  i n the f i e l d s  Moran 1936;  Morrison 1948)  f o r many p r a c t i c a l  of Animal Husbandry  This subject has a l r e a d y been r e -  viewed by others (Steenbock 1924;  I t i s worthy of mention  et a l . , 1924;  Jacobs 1944;  Heyroth  and hence i t i s f e l t  Hess and Weinstock  1947;  Manard  1947;  that a r e p e t i t i o n of  these reviews need not be i n c l u d e d i n t h i s r e p o r t , although i t has been c a r r i e d out as background m a t e r i a l .  -  3  -  L i t e r a t u r e Review A.  Action of U l t r a v i o l e t L i g h t To-day i t  on B a c t e r i a l G r o w t h  i s g e n e r a l l y accepted  that nearly  b a c t e r i a may be k i l l e d  or a t t e n u a t e d by u l t r a v i o l e t  of  but t h a t  c e r t a i n wavelengths  siderably in their been s u g g e s t e d with the  s u s c e p t a b i l i t y to  that  a n i m a l body are  solar  The l a t t e r  species  energy  vary con-  destruction.  It  those organisms which occur i n  t h o s e whose e n v i r o n m e n t 1946).  different  all  has  association  g e n e r a l l y more e a s i l y k i l l e d is flooded with  sunlight  may become more a d a p t e d t o  energy and p a r t i c u l a r l y t o i t s  than  (Luckiesh,  exposure  ultraviolet  to  component.  (Figure 1). The wave l e n g t h r a n g e 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 b e e n t e r m e d as F i g u r e 2 .  g e r m i c i d a l curve presented the  It  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 extends  the  may be n o t e d over the  that  wave-  §  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 a n d 2662 A . G a t e s (1929) n o t e d t h a t t h e w a v e l e n g t h o f lengths  maximum l e t h a l e f f e c t i v e n e s s bacteria  irradiated.  He f o u n d t h a t t h e  l e n g t h f o r the for  E. c o l i . It  and L a v i n  v a r i e s w i t h the  destruction o 2537 A .  is interesting  of Staph,  species  of  most  e f f e c t i v e waveo a u r e u s was 2650 A a n d  to mention the work of Smithburn  (1937) i n c o n n e c t i o n w i t h  Mycobacterium t u b e r c u l o s i s .  the  i r r a d i a t i o n of  They f o u n d t h a t w i t h s u b l e t h a l o d o s e s o f m o n o c h r o m a t i c 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  UltraViolet  53000 A  Infra-Red  .... 3.14 x 10 2  x-rays y-rays  H e r t z i a n Waves^ Radio Waves )  " 9000 _J 1  1 — g  10,000 R Wave Length I :  Antirachitic Radiation FIGURE I .  Chart Showing R e l a t i o n of Type and Wave Length of Radiant Energy (Hawk, et a l . 1947) ( F i g u r e s i n d i c a t e wave l e n g t h s ( A ) expressed i n A  Footnote:  One micron (JJL) - 0.001 m$. One m i l l i m i c r o n (mu) - 0.001 u One Angstom u n i t (A) - 0.1 nyu.  0  i n v i r u l e n c e and f i n a l l y troyed.  became a v i r u l e n t w i t h o u t b e i n g  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  immunity w h i l e those organisms  of the  k i l l e d by the  were n o t  a measurable  same w a v e l e n g t h s  immunity i n v a c c i n a t e d  S  1  0  Middle TC  CO  H ©  Near  ffTtn,  0  7  80 > •H  of inducing  Energy  Far rc^rmicidal  were  animals.  Ultraviolet  ^  induce  same s t r a i n t h a t capable  des-  \  6 0  40  \  20 0 2000  2400  *  4*  \  2800  3200  Angstrom U n i t s FIGURE 2 :  The above r e s u l t s  G e r m i c i d a l Curve . (General E l e c t r i c B u l l e t i n ,  w e r e o b t a i n e d when b a c t e r i a l  were e x p o s 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 . 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 an e f f e c t  on c u l t u r e m e d i a .  gels  cells  been  i f ultraviolet irradiation  B l a n k and A r n o l d o  that u l t r a v i o l e t i r r a d i a t i o n (at water  T h e r e have  1947).  (1935)  2537 A ) o f a g a r  so a l t e r e i c u l t u r e m e d i a s u b s e q u e n t l y  or  has  showed  agar-  made f r o m  them t h a t t h e y w i l l Baumgartner ion  a shift  that the  changes  before for  the  o c c u r r e d as  this  i n o c u l a t i o n the  normal b a c t e r i a l  of  u l t r a v i o l e t energy 1943.X  It  was  ^ alcohols, e v i d e n c e was  If plates  presented  were i r r a d i a t e d t o the  ^  aldehydes,  proper  and level  support  of b a c t e r i o l o g y devote  some s p a c e  b a c t e r i c i d a l or b a c t e r i o s t a t i c ( H e y r o t h , i941j S a l l e ,  been o f f e r e d .  to  action  1943;; T o p l e y a n d  g e r m i c i d a l r a n g e o f 2000 t o  established  for a considerable  no c o m p l e t e l y a d e q u a t e e x p l a n a t i o n o f t h e m e c h a n i s m  been a d v a n c e d  suggested  polysaccharides  pH was r e a d j u s t e d  Although the  2950 A has been f i r m l y  has y e t  brings  growth.  d e s c r i p t i o n of the  Wilson,  follows:  irradiat-  solutions  o r g a n i s m t h e n t h e medium w o u l d  Most t e x t b o o k s the  shown t h a t  greater a c i d i t y .  No e x p e r i m e n t a l  contention.  particular  growth of B . s u b t i l i s .  (1936) have  ^ monosaccharides  and a c i d s .  to support  the  m e d i a and c a r b o h y d r a t e  i n pH t o w a r d s  disaccharides ketones  support  (1936) and P r a t t  of carbohydrate  about  not  A number o f w o r k i n g h y p o t h e s e s  ( H e y r o t h , 1941J S a l l e ,  1943).  time  involved have  B.  A Hypothesis Proposed t o E x p l a i n the E f f e c t s . 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 m e n t i o n e d e a r l i e r a new c o n c e p t  iostasis this  due t o i n i m i c a l a g e n t s has  recent  concept  reactions,  effective  w i t h an " e s s e n t i a l  as  a result  Fildes  of b a c t e r i a r e s u l t s  metabolite". 1942) t h a t result  altered  1  i n the  It  inhibitors  of t h e i r  inter-  t h e o r y to. t h e g e r m i c i d a l  production of a with  some  i s known, i n c e r t a i n c a s e s at  absorption i n the  of r a d i a n t  the  sub-  "essential  least,  (Morton,  e n e r g y b y a c h e m i c a l compound  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  chemical configuration.  on e r g o s t e r o l ,  The a c t i o n o f u l t r a v i o l e t  f o r example r e s u l t s  a new compound, n a m e l y c a l c i f e r o l  £  block  metabolite"^  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  light  are  of u l t r a v i o l e t r a d i a t i o n suggests immediately that  irradiation  will  bacter-  Briefly  a b i l i t y to  F i l d e s (1940) p r o p o s e d t h a t  The a p p l i c a t i o n o f t h e effect  to explain  been p r o p o s e d .  a c t i v i t y to t h e i r  of b a c t e r i a l growth are ference  Ultra-  suggests t h a t c e r t a i n agents which  b a c t e r i c i d a l owe t h e i r essential  of  i n the  formation of  ( 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 substance which takes part i n a chain of syntheses necessary for b a c t e r i a l growth, A " g r o w t h f a c t o r " on t h e o t h e r h a n d , 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 a n d 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 cannot s y n t h e s i z e .  - 8•  TACHYSTEROL  With t h i s  CALCIFEROL  information at  hand i t  does n o t  (D > 2  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 c a u s e a change likely  i n some " e s s e n t i a l  metabolite".  t o be so s l i g h t t h a t t h e  iently, like  its  non-irradiated  precursor  i n a c e l l u l a r system i n p l a c e  However,  at t h e  that the  new compound w i l l  by t h i s  metabolite.  is  new compound f o r m e d i s  bination  same t i m e t h e  T h i s change  suffic-  t o e n t e r i n t o com-  of t h e normal  metabolite.  c h a n g e may be s u f f i c i e n t l y g r e a t  fail  t o meet t h e  function  S u c h a compound s h o u l d p o s s e s s  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  that  of the  served an  most  germ-  -  9 -  icidal  portion of u l t r a v i o l e t i r r a d i a t i o n .  pounds  possessing  at  well established  t h e same t i m e p o s s e s s i n g  c i d a l wavelength range are  metabolic  absorption the  and p h e n y l a l a n i n e ,  reported  t h a t the  aromatic  substances i n b a c t e r i a ultraviolet workers  light.  phenylalanine provided  affected  In a d d i t i o n to t h i s ultraviolet not  the  absorption  are  by t h e  among  absorbed  the  a c t i o n of  information  these  produced  by t y r o s i n e  K o b e r (1915)  by  and  has  H a r r i s and H o y t ' s work by  band f o r t y r o s i n e  2480 and 2970 1 a n d t h a t f o r p h e n y l a l a n i n e 2710  benzene.  tryptophane,  radiations  r e l a t i v e l y non t o x i c .  i n d i r e c t confirmation for  showing t h a t  germi-  H a r r i s a n d Hoyt (1919)  amino a c i d r a d i c a l s  of wavelengths  are  of  and  a l l p o s s e s s w e l l m a r k e d maxima  t h a t are  c o n c l u d e t h a t the  the mercury arc  maxima i n t h e  amino a c i d s ,  r a n g e o f 2960 t o 2100 A .  w i t h i n the  functions  para d e r i v a t i v e s  P - a m i n o b e n z o i c a c i d (PABA) and t h e tyrosine  Among t h o s e c o m -  lies  between  between 2360 a n d  8. The a b s o r p t i o n  w h i l e the 3100 A . ability  to  absorb  that l i e well  i s the  i n the  suggestion  length range,  case,  As f a r  as  it  amine has  of u l t r a v i o l e t  due t o t h e  the  energy  i s known t h e r e has  t h a t the g e r m i c i d a l e f f e c t o  2960 t o 2100 A , i s  properties.  aromatic  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 as  PABA t o a compound t h a t w i l l iostatic  this  those wavelengths  t r a t e d i n Figure 2. previous  2785 X ( F i g u r e 3 )  band e x t e n d s o v e r t h e r a n g e o f 2350 t o  absorbing Since this  maximum o f PABA i s a t  been  of the  alteration  possess b a c t e r i c i d a l or  illusno  waveof bacter-  - 10 -  23 24  26 27 28 29 30 31 32 33  2 5  W a v e - L e n g t h (X x Figure 3.  C.  10  )  U l t r a v i o l e t A b s o r p t i o n Spectrum of p-Amino Benzoic A c i d (0.00005 m o l a r )  Enlargement of the Working Hypothesis t o E x p l a i n Effects  of 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  P r e v i o u s l y i t was s u g g e s t e d irradiation so a l t e r  that  o f an " e s s e n t i a l m e t a b o l i t e "  that metabolite  that  it  the  ultraviolet  (eg.  PABA) m i g h t serve  its  In his recent  on b a c t e r i a l m e t a b o l i s m , Woods ( 1 9 4 7 ) p r o p o s e s the  the  Metabolism.  c o u l d no l o n g e r  normal f u n c t i o n i n c e l l u l a r metabolism.  of  - 2  review  t h a t the  f u n c t i o n a l m e c h a n i c s o f an " e s s e n t i a l m e t a b o l i t e "  n o t c o m p l e t e d when i t s He c o n s i d e r s cellular  that  processes  c e r n e d a n d on t h e  it  chemical i d e n t i f i c a t i o n i s  is essential  i n w h i c h the basis  of t h i s  to unfold  factor has  the  is  established.  precise  or f a c t o r s  enumerated  study  are  five  conapproaches  -  to the  general  problem.  11 -  They  are:  ( 1 ) To d e m o n s t r a t e t h a t a component o f an I s o l a t e d enzyme s y s t e m 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 e s s e n t i a l m e t a bolite". 11  ( 2 ) To s t u d y t h e m e t a b o l i s m o f o r g a n i s m s grown on m e d i a d e f i c i e n t i n t h e f a c t o r i n question. 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 of 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 e m p l o y 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 t h e 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 the u t i l i z a t i o n of the factor. (4) To s t u d y t h e 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 processes of the c e l l . ( 5 ) To d i s c o v e r , i n g r o w t h e x p e r i m e n t s , 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) of 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 t h e 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 of (X) or v i c e - v e r s a . The above f i v e  approaches i n the  o l i s m of growth f a c t o r s o u t l i n e d by Woods It in  (Rothman a n d R u b i n ,  as t h e acid  1942).  ultimate  bacteriostatic  i s reduced,  as  molecular  presence of  With t h i s  PABA i s t h e  on t h e  resulting  i n f o r m a t i o n i t may be reactions acid  When p - n i t r o b e n z o i c product.  ( 1 9 3 6 ) made some  nature of the  change  oxygen  production of p - n i t r o b e n z o i c  anti-metabolite.  M a l a v i y a and D u t t observations  the  t h a t the  on i r r a d i a t i o n o f PABA a c h a i n o f  occurs w i t h the  metab-  (1947).  has a l r e a d y b e e n r e p o r t e d  that  study o f the  have b e e n u s e d by many w o r k e r s  PABA on i r r a d i a t i o n r e q u i r e s  possible  a  products  quantitative formed by  the  -  12  p r o l o n g e d exposure t o t r o p i c a l compounds. phenazine not  - 1,5 but  sodium s a l t  the  of p,  para  aminobenzoic  of  organic  a c i d gave  meta compound  did  acid i n alkaline  s o l u t i o n gave  the  p^-azobenzene-dicarboxylic future  a c i d or p,  bacteriostatic  o f a number  the  chain of r e a c t i o n s  p-nitrobenzoic  ortho  - dicarboxylic acid,  As a g u i d e t o  the  sunlight  They n o t i c e d t h a t  alter,  following  -  work i t  is  acid.  hypothesized  may o c c u r w i t h t h e  that  the  production  p^-azobenzene-dicarboxylic  acid  of as  anti-metabolite.  N=0 p-hydroxylamine benzoic a c i d  p-aminobenzoic acid  p-nitrobenzoic acid  p-nitrosobenzoic acid  or:  ^  COOH-<^^>-  NHHH-^ZJ^ p,p  COOH  -<^ZT^>-  COOH  *  HgO  -hydrazobenzene—dicarboxylic acid  NaN-^.  ^  COOH  •  H 0 2  p,p^-azobenzene-dicarboxylic acid  -  It produced similar  13  must be a d m i t t e d ,  i n the  however,  PABA m o l e c u l e b y u l t r a v i o l e t  and t y r o s i n e ,  alterations  e n e r g y may be  h a v i n g an a b s o r p t i o n  t w e e n 2400 and 2900 X ( H e i d t , light.  1936) are  1926).  The n o n - a r o m a t i c  alanine,  valine,  leucine  and a s p a r t i c  that after  to  1941).  H e n r i and h i s a s s o c i a t e s  i r r a d i a t i o n by a m e r c u r y a r c  for  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  to 7.45.  They c o n c l u d e d t h a t  formation  o f ammonia as a r e s u l t NH CH C00H 2  «.  2  ultra-  influence  s u c h as  a c i d show no  o r show i t  less  glycine,  increased readily  (1934)  observed  fifteen  hours,  H 0 •  the  hydrolysis:  •>  2  a  i n pH f r o m 4 . 8  t h i s c h a n g e was due t o of  acids.  maximum b e -  sensitive  amino a c i d s  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 (Heyroth,  amino  They c o m b i n e w i t h o x y g e n u n d e r i t s  (Harris,  0.1  that the  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 t h e  Tryptophane  violet  -  H0CH C00H  * NH3  2  glycine (•0-amino a c e t i c  acid)  (<-hydroxy  acetic  acid)  The a b o v e 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 t h e chemistry is the  o f PABA.  subjected formation  It  may be t h a t ,  to u l t r a v i o l e t  when t h i s  radiations,  of p-hydroxybenzoic  it  a c i d and  aromatic  photoamine  i s hydrolyzed with ammonia:  C00H f NH3  The a b i l i t y o f u l t r a v i o l e t bactericidal  or b a c t e r i o s t a t i c  by many w o r k e r s  radiations  effects  as a p h o t o c h e m i c a l  has  process  to  been  produce recognized  (Heyroth,  1941J  - 14  Gl asstone,  1948),  -  and c o n s e q u e n t l y i t  i s important to  review  t h e modern v i e w s on p h o t o c h e m i c a l p h e n o m e n a . A brief this report  D.  as A p p e n d i x  I,  p-Aminobenzoic Acid: If  of  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  mode o f A c t i o n o f S u l p h o n a m i d e s .  an a n t i - m e t a b o l i t e  PABA t h e m e c h a n i s m o f i t s  proposed to e x p l a i n the  that  Fildes  it  i s necessary  of the  t h a t the  used t h i s the  sulphonamides.  illustrate suggested  the  t h a t the  b l o c k i n g the  the  It  it.  of the  Here,  inimical  I n 1940 Woods  sulphonamides,  hypothesis.  He has  because of t h e i r  "essential metabolite"  structural  PABA, f u n c t i o n b y  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 hypothesis by the  sulphonamides are b a c t e r i o s t a t i c  and t h a t . t h e i r  a  has s i n c e b e e n u s e d e x t e n s i v e l y t o  Woods was l e d t o t h i s  l a r g e amounts  for  antibacterial a c t i v i t y of  "essential metabolite"  s i m i l a r i t y to the  pound.  enzyme w h i c h a c t i v a t e s  of the m e t a b o l i t e .  con-  by c o m b i n i n g  or competing w i t h i t  concept to e x p l a i n the  that  sulphonamides.  either  molecular structure  agent s h o u l d resemble t h a t  to  (1940) put f o r t h the  a g e n t s may a c t  w i t h an " e s s e n t i a l m e t a b o l i t e " surface  irradiation  i n h i b i t o r y a c t i o n of the  chemotherapeutic  p l a c e on t h e  formed from the  a c t i o n may be s i m i l a r  As was p r e v i o u s l y m e n t i o n e d , cept  is  that  rather than b a c t e r i c i d a l  a c t i o n c a n be r e v e r s e d b y t h e o f PABA t o t h e  fact  com-  a d d i t i o n of  sulphonamide poisoned  cell  -  system.-  H i s work i s  He r e p o r t e d  the  bacteriostatic  c o m p e t i t i v e l y by PABA.  substances are, related  given i n greater  that the  was r e v e r s e d  15  as has  structurally,  latter  since they  (Table  of  its  Woods ( 1 9 4 0 ) of  s u l p h a n i l a m i d e on t h e  antagonized the  demonstrated  latter  b y PABA.  can n e u t r a l i z e  25,000 molecules o f t h e were a c t i v e exception  product  very closely that  suggested  that the  that  in  p o i n t by  of t h e  the virtue  reaction.  inhibitory action haemolyticus  show t h a t  Substances  concentrations  whose  antagonistic  involved  was  one m o l e c u l e o f  the b a c t e r i o s t a t i c  former.  only i n higher  of novocaine,  was t h e n  growth of S t r e p t .  His r e s u l t s  sulphanilamide  i s r e p l a c e d by a c a r b o x y l  d o e s so a t t h i s  to the  point.  only i n the f a c t  enzyme r e a c t i o n  o f PABA a n d t h a t i t  chemical s i m i l a r i t y  It  this  These two  differ  former  III).  sulphanilamide i n h i b i t s the synthesis  action of  been m e n t i o n e d b e f o r e ,  sulphonamide group of the  group i n t h e  d e t a i l at  action related  o f 500 to  PABA  (Table I) with  a c t i v i t y approaches that  to  the  of  PABA. The r e v e r s a b i l i t y o f t h e PABA s u g g e s t e d  that t h i s aromatic  a c t i o n of s u l p h a n i l a m i d e amine  is  b a c t e r i a l m e t a b o l i s m a n d i t was p r e d i c t e d would appear at  some t i m e as  Rubbo and G i l l e s p i e  (1940)  compound w a s n e c e s s a r y After  this discovery,  more t h a n t w e n t y - f i v e  a bacterial  i n the  for the Rubbo e t organic  important  in  by Woods t h a t growth  same y e a r  by  it  factor.  found t h a t  this  growth of C I . a c e t o b u t y l i c u m . a l . (1941) compounds  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  tested a series  i n o r d e r t o show  and g r o w t h  of the  stimulation.  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 of Substances 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 ) . Concentration  of s u l p h a n i l a m i d e  Substance  = 3.03 x  10~^M.  A c t i v e at M cone.  p-Aminobenzoic A c i d o-Aminoberizoic A c i d m-Aminobenzoic A c i d p-Nitrobenzoic Acid Ethyl-p-aminobenzoate Novocaine p-Hydroxybenzoic a c i d p-Toluic acid Benzoic Acid Benzamide p-Aminobenzamide 2-(p-Aminobenzylamins) p-Aminophenol Sulphanilic acid  1.2 - 5.8 x 1 0 " 0.9 1.8 3.6 5.8  x x x x  -  8  10"3 10-4 10-5 10~ 8  mm mm  1.4 x 1 0 " 0.9 x 1 0 3 6  pyridine  -  +  - i n d i c a t e s substance i n a c t i v e at 10"3M •+• i n h i b i t s g r o w t h down t o 3 . 6 x 1 0 - 5 M + i n h i b i t s growth at.10~3M  The r e s u l t s  of t h e i r  These w o r k e r s  experiments  observed  stimulating properties di-substituted lr4  positions.  importance, the  that  are tabulated  t h e compounds  are aromatic  i n Table I I *  possessing  compounds  growth  containing a  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 The o r i e n t a t i o n  a p p e a r s t o be o f  and any d e p a r t u r e s from i t r e d u c e s  a c t i v i t y o f t h e compound.  The g r e a t e s t  occupy  fundamental  considerably  a c t i v i t y i s shown  when one o f t h e two i s an a m i n o g r o u p a n d t h e o t h e r  a carboxyl  -  or c a r b o x y m e t h y l  is  o r by - OH, o r t h e  CH2OH,  -  N(CH^.)2  1:4  sub-  active  and  substituted  i n complete  of  the  r i n g o f PABA by  of t h e  amino g r o u p  i n a c t i v a t i o n of  by  the  derivative. has  when e x p o s e d  been s u g g e s t e d e a r l i e r  to u l t r a v i o l e t  occur w i t h the benzoic  formation  in this  radiations  o f a new s u b s t a n c e ,  I and I I ,  reverse the  haemolyticus  and n o t i c e  It  In a d d i t i o n , ' i t  ostatic  action  of s u l p h a n i l a m i d e  as a g r o w t h f a c t o r  be c o n c l u d e d a t t h i s  point  i r r a d i a t i o n product  may be a r e l a t i o n s h i p  interesting  (Table  (Table  I)  bacteri-  appears  However, i t  o f PABA on t h i s  basis  a c i d to the  possible that  is also  a s e r i e s of r e a c t i o n s similar  because  described the  w i t h the  compounds.  to  cannot  that p-hydroxybenzoic a c i d i s  of p - n i t r o b e n z p i c  of s t r u c t u r a l l y  Strept.  CI.  the  and a l s o  II).  and D u t t  It  on  acid  p-hydroxy-  of reversing  a c i d , as  number  very  p-nitro-  a c t i v i t y for  p,p^"-azobenzene-dicarboxylic  PABA p r o d u c e s  is  i s noted that  a c i d a p p e a r s t o be i n c a p a b l e  (1936).  namely  that p-nitrobenzoic  growth f a c t o r  benzoic  be i n a c t i v e  that  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  and a l s o has  acetobutvlicum.  report  a c h a n g e o f PABA may  a c i d or p - h y d r o x y b e n z o i c a c i d .  to study Tables  sole  amino  The r e p l a c e m e n t  replacement  ° r - OH, r e s u l t s  It  will  by c o m p a r i n g t h e  given i n Table I I .  c a r b o x y l group i n the  -  new  o f th.e c a r b o x y l a n d t h e  best i l l u s t r a t e d  i n a c t i v e compounds of t h e  -  group.  The i m p o r t a n c e stituents  17  the  there  substance,  by M a l a v i y a irradiation  formation  of  of a  - 18  TABLE  -  II.  G r o w t h 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 Related 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  Substance p-Aminophenyl a c e t i c a c i d p-Aminobenzoic a c i d p - A m i n o b e n z o i c a c i d (Na e s t e r ) p-Aminobenzoic a c i d ( e t h y l e s t e r ) p-Aminobenzaldehyde p-Aminobenzoic a c i d (benzoyl derivative) p-Aminobenzoic a c i d (diethylaminoethyl ester) p-Nitrobenzoic acid p - N i t r o ' benzaldehyde p-Methyl benzoic a c i d o-Aminobenzoic a c i d m-Aminobenzoic a c i d p-Aminobenzamide p-Aminobenzyl a l c o h o l p-Hydroxy benzoic a c i d p-Aminophenol p-Dimethylaminobenzaldehyde p-Aminobenzene sulphonamide Benzene Benzoic a c i d Inositol Nicotinic acid Thiamin Tryptophane Tyrosine Cholesterol  Growth  +  * t  + +  -  Growth F a c t o r Activity* 1 unit in 0.00001 0.0001 0.0001 0.0001 0.0001 0.001  ug. ug. Ug. /lg. ug. ug.  0.001  •ug.  0.001 0.01 1.0 10.0 10.0 10.0  /ig. ug. ug. >lg. ug. Jig.  —  --  -mm mm  mm  —  mm  mm mm  -  mmmm  mm  mm  mm mm  —  -mm mm  —  mm mm 7  x G r o w t h 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 growth of C I . aicetob u t y l i c u m i n 5 m l . o f b a s a l medium u n d e r 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 .  -  E.  Relation ~ The  19  -  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 >  bacteriostatic  mechanism o f s u l p h a n i l a m i d e p r o -  p o s e d b y Woods ( 1 9 4 0 ) a n d F i l d e s l i s h e d by subsequent w o r k e r s Lampen a n d P e t e r s o n , investigators to t h e i r  Not it  is  1940;  1 9 4 1 ; L a n d y a n d Wyeno, 1 9 4 1 ) .  Some  have c o r r e l a t e d t h e  a l , 1942), while r e l a t e d t o the  that  a c t i v i t y of  sulphonamides  ( F o x and R o s e , 1942;  o t h e r s seem t o s u g g e s t t h a t  basic d i s s o c i a t i o n constant  o n l y has PABA a s t e r i c a l s o has  estab-  (Rubbo and G i l l e s p i e ,  acid d i s s o c i a t i o n constant  Schmelkes et activity  ( 1 9 4 0 ) has been w e l l  (pK ). b  s i m i l a r i t y to sulphonamides,  a characteristic ability  t o form s a l t s  the.  but  similar  to  drug. B e l l and R o b l i n  chemical properties  ( 1 9 4 2 ) made a s t u d y o f t h e  of s u l p h a n i l a m i d e .  the  bacteriostatic  may  be c o r r e l a t e d w i t h t h e  g r o u p and w i t h t h e investigators  the  character  acid d i s s o c i a t i o n constant.  that  compounds  of t h e S O 2 These  p o i n t e d o u t t h a t t h e more n e g a t i v e t h e S O 2  g r o u p t h e more a c t i v e t h e negative  They s u g g e s t e d  a c t i v i t y of s u l p h a n i l a m i d e type negative  physico-  S 0  g r o u p i n PABA a t  2  compound w i l l  group i s ,  pH %.  be,  and a l s o t h e  t h e more i t r e s e m b l e s  A t pH 7 t h e  the C O 2  c a r b o x y l g r o u p i n PABA i s  over n i n e t y - n i n e percent  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  group c a r r i e s  charge.  crease the  a negative  b a s i c i t y of the  Bell  p a r a amino r a d i c a l t h r e e - f o l d  and R o b l i n  2  The c a r b o x y l i o n may i n -  the value obtained w i t h u n - i o n i z e d carboxyl With these data.  more  over  i n an a c i d m e d i u m .  (1942) p r e d i c t e d the  relative  -  a c t i v i t y o f the  -  2 0  i o n i c and m o l e c u l a r forms of t h e  amide t y p e d r u g s .  S c h m e l k e s a n d Wyss (1942) o b s e r v e d  the a n t i - b a c t e r i a l e f f i c i e n c y a b l e by i t s pH o f t h e  sulphanil-  o f a s u l p h o n a m i d e as  that  measur-  a b i l i t y t o overcome PABA was d e p e n d e n t upon  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  more f a v o r a b l y w i t h PABA a t  a high r a t h e r than at  compete 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 weaker a c i d . the  K u m l e r a n d H a l v e r s t a d t (1941) s u g g e s t e d  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  w i t h the  c o n t r i b u t i o n of the  t i o n o f the  resonating  the  a that  associated  form w i t h a  separa-  charge: H  0  R  N 0  The  o b s e r v a t i o n made b y K u m l e r a n d D a n i e l s (1943)  indicates that t i v e charge of  any e f f e c t t h a t  on t h e  S O 2 group w i l l  t h i s form with a separation  t h a t the  bacteriostatic  negative  character  idea that  the  increases the increase  of c h a r g e .  a c t i v i t y is  nega-  contribution  The o b s e r v a t i o n the  SO^ g r o u p i s c o m p a t i b l e w i t h associated with of charge.  the  the  resonating  The n e g a t i v i t y o f  S O 2 g r o u p and t h e c o n t r i b u t i o n o f t h e separation  the  a c t i v i t y i s associated with  of the  form h a v i n g a s e p a r a t i o n  relative  the  r e s o n a t i n g form w i t h  of charge are concomitant f a c t o r s  r e l a t i n g to  one  another. 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 separation  o f charge  increases  the  amino g r o u p i s  a  affected  a  -  three ways.  i t t a k e s on a p l u s  double bonded t o c a r b o n ,  structure  amino g r o u p t h a t  it  i s these p r o p e r t i e s  is chiefly  of the  of the  r e s p o n s i b l e f o r the  S O 2 group.  c o n c e p t w e r e as f o l l o w s . sulphonamides  a c t i v i t y of  negative  In the  first  place the  while  on t h e  other  are  influence through the  part  one.  exert  their  by p l a c i n g d i f f e r e n t from the  R groups  effect  functional in activity  on t he s u l p h o n -  t h e R g r o u p has  and i n d u c t i o n (Kumler  on  the  and  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 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 , s o l u t i o n s by Kumler a n d S t r a i g h t developed i n that the (unsubstituted acid  the  p-amino group r a t h e r than the S O 2  amino group t h r o u g h r e s o n a n c e Daniels,  the  e s s e n t i a l l y c h e m i c a l , a n d hence  o f t h e m o l e c u l e f o r a c t i v i t y and c h a n g e s  amide g r o u p r e s u l t s  enters  The r e a c t i o n s .  appears t h a t t h e amino group i s t h e  brought about  S O 2 group;  hand,  c h e m i c a l l y r e a c t i v e c o m p o n e n t s w o u l d more l i k e l y  It  para  The r e a s o n s f o r p r o p o s i n g t h i s  a r o m a t i c amino group i s a v e r y r e a c t i v e  group.  it  i s n o t an a c t i v e g r o u p c h e m i c a l l y ; i t  i n t o a l m o s t no r e a c t i o n s ,  of enzyme systems  and  Kumler and D a n i e l s  s u l p h a n i l a m i d e t y p e compounds a n d n o t t h e  character  in  charge;  resulting in a quinoidal  ( K u m l e r a n d D a n i e l s , 1943).  (1943) b e l i e v e t h a t  the  -  The a m i n o g r o u p has a t e n d e n c y t o become co?<-  planar w i t h t h e r i n g ; becomes  2 1  (strong  b a s i c and w a t e r  (1943).  A general rule  s p e c t r u m o f any a r o m a t i c  to the  salt)  was  amino  o r a l k y l d e r i v a t i v e ) compound w i l l  enough t o c o n v e r t i t  benzene  to  revert the  in  -  spectrum of the  2  2 -  c o r r e s p o n d i n g compound i n w h i c h t h e  g r o u p i s r e p l a c e d by h y d r o g e n o r an a l k y l amide and PABA have a h i g h e r  in  the  i o n than  i n the  solution.  This  form makes a g r e a t e r  basic  indicates  contribution  undissociated molecule.  K u m l e r and H a l v e r s t a d t a n d s u l p h o n a m i d e s have s i m i l a r a separation  Sulphanil-  extinction coefficient in  than i n sodium c h l o r i d e or water t h a t the main resonance  group.  amino  of charge the  ( 1 9 4 1 ) showed t h a t resonating  therapeutic  since  structures  PABA  with  p o t e n c y may be  assoc-  i a t e d o n l y w i t h f o r m s w h i c h have s u c h a. c h a r g e .  F.  Other N u t r i t i o n a l P u r s u i n g the  in  efforts  to  Antagonistic Reactions  h y p o t h e s i s o f Woods, o t h e r  produce b a c t e r i o s t a t i c  other vitamins s t r u c t u r a l in  compounds,  presented.  The c o n c e p t t h a t  may  Y e t even more s p e c t a c u l a r  p r o t e i n d e r i v a t i v e s and other  a c t u a l l y induce the  lack of essential Mc I l w a i n acid  a  or  was s t a r t l i n g e n o u g h when i s the  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 vitamins,  to  involved  may be n e g a t i v e l y p r o d u c e d b y t h e r e d u c t i o n  omission of v i t a m i n s i n the d i e t first  applied  changes s i m i l a r . t o t h o s e  p a s s i n g f r o m PABA t o s u l p h a n i l a m i d e .  disease  investigators,  fact  essential  important  metabolites  deficiency signs associated with  the  nutrilites. (1940) found t h a t  pyridine - 3 -  sulphonic  i n h i b i t e d b a c t e r i a l growth i n c o m p e t i t i o n with  nicotinic  - 23  a c i d and t h a t  -  <*( - a m i n o s u l p h o n i c a c i d s  competition with^-amino acids Snell  (1941)  panic  acid inhibited bacterial  pantothenic of  and l a t e r  acid.  structural  produce  acted  (Mc I l w a i n ,  Mc I l w a i n ( 1 9 4 2 )  Thus f a r  similarly 1941).  Likewise  showed t h a t  thio-  growth i n c o m p e t i t i o n w i t h  t h e w o r k has  c h a n g e a n d has b e e n l i m i t e d  i n v o l v e d one by t h e  type  desire  to  o n l y i n h i b i t i o n of m i c r o b i a l growth. The n e x t  advance  was made when i t was shown  c e r t a i n compounds r e l a t e d metabolites animals.  in mice.  structurally  to vitamins  showed t h a t  production of t y p i c a l signs T h i s compound a p p a r e n t l y  m i c e on a d i e t From t h e  deficient  above  of thiamine  can produce  i n the  other  important substances are  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 , by the m e t a b o l i t e s  adequate amounts. or p r e p a r e d ,  or competitive are  can be  tabulated  deficiency  does  concluded  feeding  that  biologically  and t h a t t h e s e s i g n s  of are  i n q u e s t i o n when g i v e n i n  usually referred  inhibitors.  deficiency  cause s p e c i f i c s i g n s  Many s u c h s u b s t a n c e s have  and a r e  the  s t r u c t u r a l l y to various able to  in  vitamin.  information i t  c e r t a i n compounds r e l a t e d  compounds  or  pyrithiamine  symptoms i n m i c e more q u i c k l y and s e v e r e l y t h a n  reversed  that  produced t y p i c a l signs o f d e f i c i e n c y d i s e a s e s  Woolley a n d W h i t e ( 1 9 4 3 )  caused the  the  in  to  III.  discovered  as m e t a b o l i c  For convenience,  i n Table  been  a few o f  analogues these  TABLE I I I . Nutritional- Antagonists  Growth  Factor  H N <  p-aminobenzoic  1947).  Antagonist  COOH  2  (Gale.  H N 2  acid  Susceptible  >" S0 NH Sulphanilamide 2  CI.  2  Organism  a c e t o b u t y l i c u m and many o t h e r s Pr. v u l g a r i s  Nicotinic  Pyridine-3-sulphonic  Acid  R.CHNH .C00H C e r t a i n amino-acids  R.CHNH .S0oH Sulphonic a c i d analogues  2  HocM^t-  acid  S. S. L.  c-co-NH-cH-fChlxrtooU ck OH  Pantothenic  Pantoyl-taurine  Acid  N - e.H  A/-a  hi  NH .CH .CH .C00H (3 - a l a n i n e " HH-co 2  o i  haemolyticus lactis arabinosus Propionibacteria  Staph,  " "  aureus  aureus  Pyrithiamine  Thiamine. 2  Staph,  2  NH .CH ,CH(CH ).C00H methyl, ft-amino-propionic 2  2  ,  «  2  acid  OC cHi Hf<- co  Barbituric  Uracil  Yeast  3  Staph,  aureus  Acid  LcHori\ An  (X  th  .^w'V  T°  b  Riboflavin  S.  ^O^s^^  Mepacrine  haemolyticus  G.  p - A m i n o b e n z o i c 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 Acid i n B a c t e r i a l N u t r i t i o n The  is  recent  chemical  an e s s e n t i a l c o n s t i t u e n t  (PGA)  molecule provides  d i s c o v e r y t h a t t h e PABA m o i e t y of the  acid  an i n t e r e s t i n g e x a m p l e o f t h e g e n e r a l  a p p r o a c h p r o p o s e d b y Woods ( 1 9 4 7 ) . of t h e  pteroylglutamic  I t i s s u g g e s t e d t h a t one  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 b o l i s m i s t o a c t a s  a precursor  for the biological  PABA may be c o n s i d e r e d sense t h a t  synthesis  o f PGA a n d t h a t  an " ' e s s e n t i a l m e t a b o l i t e "  only i n the  i t i s part of a l a r g e r molecule which i s the  a c t u a l growth f a c t o r . Before there  the chemical  appeared t o e x i s t  f a c t o r a n d PABA.  nature  o f PGA was e s t a b l i s h e d  a b i o l o g i c a l r e l a t i o n between  Mayer ( 1 9 4 3 ) r e p o r t e d  that a yellow  this pig~  ment was f o r m e d i n c u l t u r e s 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 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 o f PABA.  I t was f o u n d t h a t t h i s  high  concentrations  p i g m e n t was n o t i d e n t i c a l  t o r i b o f l a v i n a n d d i d n o t a p p e a r i n t h e c u l t u r e s when no PABA was p r e s e n t . this  A yellow  p i g m e n t was a l s o p r o d u c e d b y  o r g a n i s m when i t s g r o w t h m e n s t r u u n c o n t a i n e d  concentrations  of procaine.  Mayer's l a t e r  (A d e r i v a t i v e o f P A B A ) .  work ( 1 9 4 4 ) i n d i c a t e d t h a t t h e  p i g m e n t i s f o r m e d by an e n z y m a t i c p r o c e s s a n d i s a compound d e r i v e d the  f r o m PABA b y o x i d a t i o n .  enzyme i s an o x i d a s e  specific  certain  and suggested t h a t  yellow  apparently  He assumed  that  i t may be a  enzyme, n a m e l y , P A B A - o x i d a s e , o r i t may be a g r o u p  o f enzymes s u c h 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 r e s i m i l a r t o PABA, s u c h as a n i l i n e a n d 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 to did  t h e medium f o r t h e g r o w t h o f M y c o b a c t e r i u m  added  tuberculosis  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,  1944). In  1 9 4 4 Mayer d e m 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 t h e  c o n c e n t r a t i o n o f 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  folic  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 .  Lampen a n d J o n e s ( 1 9 4 6 ) folicj acid  no f o l i c  acid  sulphonamide.  s y n t h e s i z i n g enzyme s y s t e m s  PABA.  amides the  have  t o be i n h i b i t e d  I n a d d i t i o n t o t h e a b o v e o b s e r v a t i o n s , Lampen folic  a c i d would over-ride the  e f f e c t s o f the s u l p h o n a m i d e s as w e l l as w o u l d  The a n t a g o n i s m between  competitive  to sulphon-  be no b a c t e r i o s t a t i c a c t i o n o f t h e  Jones (1946) n o t e d t h a t  bacteriostatic  are not susceptible  require:  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  and t h u s t h e r e w i l l  and  observed t h a t b a c t e r i a which  i n t h e i r media  amide i n h i b i t i o n .  Furthermore,  s u l p h o n a m i d e s a n d PABA i s  (Ansbacher, 1944), w h i l e t h a t  and f o l i c  acid i s non-competitive.  between  sulphon-  I n o t h e r words,  same amount o f PGA w o u l d overcome a l a r g e d o s e  of t h e  d r u g e q u a l l y as w e l l a s a s m a l l e r a m o u n t . It effect  w i l l be i n t e r e s t i n g t o n o t e i f t h e b a c t e r i o s t a t i c  ( 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  n o r m a l PABA i s -c o m p e t i t i v e o r n o n - c o m p e t i t i v e .  I t does  l i k e l y t h a t t h e f o r m e r p r o d u c t w o u l d be c o m p e t i n g w i t h for  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 .  seem PABA  I f the irradiated  -  27 -  p r o d u c t c a n b l o c k t h e enzyme s y s t e m so t h a t n o r m a l  PABA  c a n n o t be u t i l i z e d b y t h e c e l l , t h e n , t h e o r e t i c a l l y t h e bacterial of the  cell  will  be i n h i b i t e d u n t i l  a proper  concentration  PABA i s a v a i l a b l e t o o v e r - r i d e t h e " b l o c k i n g e f f e c t " o f i r r a d i a t i o n product. D u r i n g 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 w o r k has been  reported to  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 s u b s t a n c e s  related  PABA ( W i l l i a m s , 1 9 4 4 ; M a r t i n a n d R o s e , 1 9 4 5 ; Lampen and  Jones, 1946a, 1947; S a r e t t , 1947).  to PABA ( M a r t i n and R o s e , 1 9 4 5 ) ,  substances r e l a t e d  them were f o u n d t o be i n h i b i t o r y , 3-chloro-4-aminobenzoic acid, Lampen and J o n e s acid,  Of t h e t h i r t y - t h r e e  (1947)  pteroic acid,  tested  three of  namely, 3-hydroxy- and  and 3 : 4 - d i a m i n o b e n z o i c a c i d .  showed t h a t  p-aminobenzoylglutamic  PGA and p t e r o y l t r i g l u t a m i c  l e s s a c t i v e on a m o l a r b a s i s t h a n i s PABA.  acid are a l l  They c o n c l u d e  by p o i n t i n g o u t t h a t PGA, p u r i n e s and t h y m i n e a r e p r o d u c t s of  enzyme s y s t e m s  i n w h i c h PABA f u n c t i o n s .  presented i n t h e i r of  PABA and r e l a t e d  report which  A d i a g r a m was  shows t h e p o s s i b l e  compounds i n b a c t e r i a l  relations  metabolism  (Figure 4 ) . PGA, i t i s c l a i m e d (Lampen a n d J o n e s , 1 9 4 7 ) i s s y n t h e s i z e d f r o m PABA and t h i s s y n t h e s i s a d d i t i o n of sulphonamides.  i s inhibited  P-aminobenzoyl-L-glutamic  (PABG) o r 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 b u t t h e r e i s no e v i d e n c e t o s u p p o r t t h i s PABG i s u t i l i z e d (Reaction I ) .  f o r growth  only a f t e r  R e a c t i o n 2 shows t h a t  statement.  by t h e acid  synthesis, However  d e g r a d a t i o n o f PABA  pteroyl-triglutamic  acid  - 28 -  can  be h y d r o l y z e d  before It  t o PGA.  This  hydrolysis i s essential  t h e t r i g l u t a m i c a c i d c a n be u t i l i z e d  by some  organisms.  3 that t h e product of t h e a c t i o n  i s suggested i n Reaction  o f PGA i s n o t t h y m i n e ( n o r t h e p u r i n e ) as s u c h , b u t 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 .  Sarett  (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  b e t w e e n PABA a n d PGA as a g r o w t h f a c t o r f o r o r g a n i s m s . reported extent  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  f o r one o r g a n i s m b u t n o t f o r a n o t h e r .  suggested that a precursor  PABA h a d o t h e r f u n c t i o n s  o f PGA and t h a t  f r e e t h e PABA b e f o r e  small  also  b e s i d e s i t s u s e as  c o n j u g a t e d PABA may have t o  utilization  t h a t compound i n i t s f r e e  He  He  form.  by o r g a n i s m s w h i c h  require  -  29 -  Growth (Amino a c i d s (Methionine ( Lysine  PABA  Purine:—-»PurineX ^ - P r e c u r s o r  Inhibited by s u l p h onamides  I n h i b i t e d by h i g h sulphon(1) amide c o n c e n trations  ••Ptefoylglutamic * acid  (2)  pt e r o y l t r i g l u tamic a c i d  Pteroic acid  PABG ( a d d e d ) Thymine-  (3)  •» ThymineX: V  Precursor  .(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 Metabolism (Lampen and J o n e s . 1947*7.  - 30 _  H.  Review  o f t h e R e l a t i o n s h i p Between p - A m i n o b e n z o i c and S u n b u r n .  Acid  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 d a t e h a s e m p h a s i z e d the r o l e  o f 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  metabolites i n relation iation  t o b a c t e r i a l growth.  anti-  That  p r o d u c t s o f PABA p r o d u c e s y s t e m i c e f f e c t s  the i r r a d -  i n animals  and man i s e v i d e n c e d by t h e e x t e n s i v e r e p o r t s w h i c h h a v e b e e n published  on t h e r e l a t i o n s h i p o f PABA t o t h e e r y t h e m a o f  sunburn.  Since the metabolism  o f mammalian c e l l s  i sprobably  s i m i l a r t o , i f not i d e n t i c a l with that 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 t h e b i o c h e m i c a l mechanism o f t h e s u n burn r e a c t i o n i s reviewed below light  i n t h e hope t h a t i t may s h e d  on t h e w o r k i n g h y p o t h e s i s p r o p o s e d f o r t h e g u i d a n c e  of t h i s  project. The p h o t o c h e m i s t r y o f t h e s u n b u r n r e a c t i o n h a s been  d i s c u s s e d b y Blum ( 1 9 4 1 ) .  The t h e o r y was a d v a n c e d  that 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 s p e c t r u m i d e n t i c a l w i t h o r 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 ) .  t h e o r y a s u b s t a n c e o r s u b s t a n c e s undergo  According t o this a photochemical  r e a c t i o n by a b s o r b i n g t h e e f f e c t i v e u l t r a v i o l e t giving rise  rays thus  t o a product o r products which i s r e s p o n s i b l e  for the biologic  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 e r y t h e m a .  A p p a r e n t l y sunburn i s caused by a narrow u l t r a v i o l e t r a y s i n t h e r e g i o n o f 2900 t o 3100 X. r e s p o n s e i n n o r m a l s k i n produced by t h i s erythema  or redding of t h e s k i n .  band o f The f i r s t  energy i s termed  T h i s may p e r s i s t f o r s e v e r a l  -  days,  a f t e r which  or t a n n i n g which subsided  3'1 -  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 may a p p e a r b e f o r e t h e e r y t h e m a h a s c o m p l e t e l y  (Blum, 1 9 4 1 ) .  to sunburn r a d i a t i o n s  The p i g m e n t w h i c h consists of granules  follows  exposure  of melanin.  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, 1 9 2 8 )  Blum ( 1 9 4 1 ) r e v i e w s this latter  s e v e r a l suggestions as t o the formation of  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 . mechanism by w h i c h  this  t r a n s f o r m a t i o n occurs i snot f u l l y  known, h o w e v e r , R a p e r ( 1 9 2 7 ) of t h e benzene n u c l e u s ring  The  suggests  that a hydroxylation  probably takes place f o l l o w e d by  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  NH  2  \  CH CH-C00H 2  \  CH CHC00H  CH CHC00H  2  2  A v  V  OH  OH  H0  H o  /\ v\  Dopa-quinone  3,4-Dihydroxyphenylalanine (Dopa)  Tyrosine  GH,  °i V /  CHCOOH  CHCOOH N H  5,6 d i h y d r o x y d i h y d r o i n d o l -<* carboxylic acid  COOH  H 5,6-Quinone (Hellachrome)  5,6-dihydro indole-.<*> carboxylic acid Melanin  -  It i s claimed formation  33  b y A n s b a c h e r (1941) t h a t PABA m o d i f i e s t h e  of melanin.  He s t a t e s t h a t PABA i s one o f a number  o f t h e more r e c e n t l y d i s c o v e r e d the process other  vitamins  dealing with the pigmentation  vitamins  found i n t h i s these  -  namely p a n t o t h e n i c category,  which f u n c t i o n s i n of the skin.  a c i d and b i o t i n  p l a y an a p p r e c i a b l e  what a p p e a r s t o be a v e r y c o m p l i c a t e d  spectra of di-3ubstituted  noted i n Figure  studying the  benzene d e r i v a t i v e s ,  compounds a b s o r b l i g h t  degree t h a n e i t h e r t h e o r t h o  part i n  problem.  M a r c h l e v s k y a n d M a y e r (1929), w h i l e  showed t h a t t h e p a r a  are a l s o  and i t i s p o s s i b l e t h a t n o t one o f  f a c t o r s , but a l l three  absorption  The  t o a much  o r meta d e r i v a t i v e s .  greater  It i s  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  lower  maxima, w i t h t h e maxima s h i f t e d t o w a r d s t h e v i s i b l e wave lengths.  T h i s work c o n f i r m s  the e a r l i e r  (1926) a n d B e h a g h e l e t a l . ( 1 9 2 8 ) , spectrographs  results  o f Rothman  i n which q u a n t i t a t i v e  methods w e r e u s e d .  Hausser and V a h l e  (1922), i n t h e c o u r s e  of studies  on t h e e r y t h e m a o f s u n b u r n , d e m o n s t r a t e d t h a t a maximum erythema e f f e c t  occurred  a t a w a v e l e n g t h o f 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 or s h o r t e r w a v e l e n g t h s . erythema producing PABA.  of e i t h e r  longer  L a t e r , H a u s s e r (1928) r e l a t e d t h i s  w a v e l e n g t h t o t h e a b s o r p t i o n maximum o f  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 t h e rays  causing the  - 3.4 -  persistent  erythema of sunburn.  laboratory  ( B e h a g h e l e t a l . , 1928) a t t e m p t e d  portion ing  of the procaine  value.  the  essential configuration  further  tecting Table  their  shown t h a t  qualities  protective  t o a s c e r t a i n the  PABA  effect.  screen-  and i t s d e r i v a t i v e s I t was  shown  that  was p a r a s u b s t i t u t i o n o f t h e groups.  I t was  t h e s u b s t i t u t i o n of t h e hydrogens of the groups  o f PABA.  d i d not a l t e r  the sunburn  The compounds s t u d i e d  IV and a r e d i v i d e d  As  that  t h e amino and c a r b o x y l i c  amine or t h e c a r b o x y l  in  revealed  f o r the - screening  benzene r i n g w i t h  work f r o m t h e same  molecule which p o s s e s s e s the  These s t u d i e s  were r e s p o n s i b l e  Later  pro-  a r e shown  i n t o two g r o u p s  on t h e b a s i s o f  the absorption  maximum o f PABA  value.  previously  stated  o i s a t 2785 A..  I t i s known,  t r y p t o p h a n e and t y r o s i n e ,  however, t h a t  have a b s o r p t i o n  proteins  maxima i n t h e r e g i o n  o f 2700 - 2800 A (Blum, 1941) and a l s o t h a t have an a b s o r p t i o n (Smith, 1929). absorption proteins  these  From t h i s  containing  amino  acids  a t 2790 and 2750 X. r e s p e c t i v e l y i t i s quite  probable  that  o f s u n b u r n r a d i a t i o n by t h e e p i d e r m i s  configuration, example  band  containing  the benzenoid  i s due t o  amino a c i d s t r u c t u r e  (Blum, 1941) a l t h o u g h  PABA, may c o n t r i b u t e  the principal  other  significantly.  i n the  substances, f o r  - 35 -  12000 11000  'S. »  10000 -  i i i  \  ;  \  i  \ \  r  \ \ \ \  1 1 1 1 1 1  9000  >  \  1 1  8000 -  »  i i \  7000  6000  5000  _  \ \  i i  »  4000  •  i  \  /  •\ I  3000  2000  i  '  1 t  ) \  \ /^  1 1 1 i \  X 1000 L *  \  2200  s  2700  /  \  \  ^ \ 3200  \  '  \ 3700  Angstrom U n i t s Figure  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 o f Aminob 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 M a y e r , 1 9 2 9 ) .  - 36 -  TABLE I V . Positive Absorb  Compounds Do. N e g a t i v e Compounds Do N o t 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).  POSITIV 1.  A  2.  A  3.  COOrl  p-Amidobenzoesaure  Anaethesin Novocainbase p-Amidobenzoesaure- p-Amidobenzoyldiaethylester aethyl-amidoaethanol  4.  5.  ./v/H-cocH  3  C06H fa)  COof^CHf^^HCt  Chlorhydrat der Novocainbase  6.  p-Dimethylaminobenzoesaure  p-Acetyl-amino benzoesaure  NEGATIV  8.  7.  9. COO-M*"  Anilin  Dimethylanilin  Benzoesaureaethyl ester  - 37 -  TABLE I V . ( 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 N o t A b s o r b 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 e t a l . 1928). NEGATIV 10.  PH  11.  CooH  p-Nitrobenzoesaure  CooH •I  V  p-Isopropylbenzoesaure  16.  15.  3  p-Aminobenzolsulfosaure  Terophtalsaure Benzol-p-Dicarbonsaure  17.  18, CH NH .C00.C H 2  2  5  Glykokollaethylester  COOH  Tyrosin p-Oxyphenyld-amino-propionsaure  AminoessigsaureAethylester  Pyridincarbon saure  21,  20.  M  /vlh.  S0 tf  COOH  2  19.  p-Methoxybenzoesaure  ij  COOH  cooU  A* COOH  p-Oxybenzoesaure  14.  13.  12.  COOti  COOH O-Aminobenzoesaure  M-Aminobenzoesaure  M-Aminobenzoesaure a e t h y l e s t e r  - 38 -  TABLE I V ( 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 N o t 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).  NEGATIV  22.  23. 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 pentanols  C h l o r h y d r a t des T e t r a methyldiamino-benzoylpentanols  - 39 -  I.  Protection  f r o m Sunburn  During the past (Bird, 1947; its  by p . A m i n o b e n z o i c  few y e a r s s u b s e q u e n t  Acid. workers  1942; Rothman and R u b i n , 1942; Rothman and Kumler  and D a n i e l s ,  derivatives  sunscreen.  shown t h a t  a p p r o a c h t h e r e q u i r e m e n t s f o r an  These  those rays that  1948) have  PABA and ideal  a r o m a t i c compounds a r e a b l e t o a b s o r b  will  and n o n - i r r i t a t i n g  Henningsen,  produce  erythema  f o r mammalian  skin.  and r e m a i n  non-toxic  - 40  -  EXPERIMENTAL I.  Introduction As  s t a t e d p r e v i o u s l y t h e p r e s e n t work was  t o t e s t a new logical  hypothesis  effect  proposes t h a t the b i o l y t i c  v i o l e t r a d i a t i o n s can such  As a s t a r t i n g  r a d i a t i o n s on c e r t a i n p o i n t i t was  has  a c t i o n of  ultra-  suggested  t h a t the  i n the  "essential  PABA s i n c e i t s a b s o r p t i o n  which might support  been r e v i e w e d  pro-  "essential metabolites".  i n t h e r a n g e o f maximum b i o l y t i c  c i r c u m s t a n t i a l evidence thesis  cells.  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  ^ m e t a b o l i t e " i n v o l v e d m i g h t w e l l be maximum l i e s  physio-  o f 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  This hypothesis  duced by  proposed to e x p l a i n the  designed  action.  such  The  a hypo-  p r e v i o u s s e c t i o n s of  this  thesis. A number o f means seemed p o s s i b l e t o d e t e r m i n e 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 , i n t h e PABA m o l e c u l e .  alteration  S u c h an a l t e r a t i o n s h o u l d be  • a b l e by p h y s i c o c h e m i c a l w o u l d be  p r o d u c e an  determinations.  of l i v i n g  the present  work.  cells.  A survey  detect-  Physiologically i t  of g r e a t e r i n t e r e s t t o demonstrate such  by t h e use  i f  alteration  B o t h methods have been u s e d i n of the  l i t e r a t u r e revealed that  a m i c r o b i o l o g i c a l method f o r t h e d e t e r m i n a t i o n o f PABA w o u l d o f f e r a number o f a d v a n t a g e s o v e r cell use  p r e p a r a t i o n s or c h e m i c a l i n recent years  assay  has  t h e use  procedures.  -The  of  other  extensive  o f 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  d e m o n s t r a t e d t h a t t h e s e methods p r o v i d e  a  - 41 -  p r e c i s i o n and a f a c i l i t y of assay not r e a d i l y o b t a i n a b l e through the use of i n t a c t animals.  A number of such micro-  b i o l o g i c a l procedures are a v a i l a b l e f o r PABA d e t e r m i n a t i o n (Appendix  II).  Appendix I I I .  Two  such methods are recorded here as  The method of Landy and Dicken (1942) u s i n g  Acetobacter suboxydans was  s e l e c t e d f o r use i n the present  work.  II.  Establishment of the Assay  Procedure;  At the outset, attempts were made t o repeat the assay procedure  of Landy and Dicken (1942) f o r PABA.  From the r e s u l t s of these p r e l i m i n a r y assays i t was that a number of p h y s i c a l and chemical f a c t o r s the response of A. suboxydans to PABA. <  an attempt carried  was  Experiment  I:  influence  For t h i s reason  made to study these f a c t o r s .  out and the r e s u l t s  evident  The  experiments  obtained are o u t l i n e d below.  Assay Procedure f o r PABA as o u t l i n e d Landy and Dicken (1942).  by  Eleven assays were c a r r i e d out f o l l o w i n g the procedure of Landy and Dicken as given i n Appendix I I I .  The  r e s u l t s of a l l these assays are presented i n Table V and a r e p r e s e n t a t i v e curve from one o f them i s g i v e n i n F i g u r e 7/.  TABLE V. The G-rowth Response of A. suboxydans to Varying Increments of PABA  Micrograms of PABA per 10 cc culture 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1  Assay 1 0.004H 0.032 0.276 0.284 0.495 0.569 0.509 0.538 0.569 0.569 0.620  Numbers  2  3  4  5  6  7  8  9  0.004 0.032 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 0.552 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  10 0.013 0.076 0.208 0.319 0.222 0.260 0.301 0.337 0.367 0.420 0.482  x Growth was measured i n the Coleman Junior Spectrophotometer at 600 mu. Figures represent growth of A. suboxydans measured as O p t i c a l Density O.D. B Log I I  s  2-log G (Galvometer r e a d i n g )  11 0.018 0.066 0.222 0.301 0.201 0.252 0.284 0.337 0.377 0.444 0.513  -  43  -  Reference t o Table V and F i g u r e 7 r e v e a l s that t h e response of  curve of the t est organism t o i n c r e a s i n g  PABA i s somewhat i r r e g u l a r ,  increments  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 o f PABA. The  response curve g i v e n by Landy and Dicken  i n d i c a t e s a smooth a n d r e g u l a r r e s p o n s e t o PABA. subsequent  n u m e r i c a l v a l u e s f o r t h e growth suboxydans their  response  present  o f PABA.  When  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 t h e  r e s p o n s e c u r v e was i r r e g u l a r ,  'It a p p e a r s  f a c t o r s m i g h t be r e s p o n s i b l e f o r t h i s  2t  In a  o b t a i n e d w i t h A.  i n t h e presence of v a r i o u s l e v e l s  d a t a were p l o t t e d  Experiment  (1943)  paper, Landy and S t r e i g h t o f f  (1942)  The E f f e c t  that  a number o f  irregularity.  o f S u r f a c e A r e a o f t h e Medium  Upon t h e G r o w t h o f A. s u b o x y d a n s . A.-suboxydans o b t a i n s i t s e n e r g y f o r g r o w t h the  under  assay 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 di-  hydroxyacetone: CH 0H  CH 0H  2  I  CHOH | CH 0H  2  dehydrogenation  _ »  2  2H  #  4- J 0  C-0  \  f 2H*  CH 0H 2  2  -~* H 0 2  S i n c e oxygen i s t h e f i n a l seemed l i k e l y  I  hydrogen  t h a t one o f t h e l i m i t i n g  acceptor i t  factors  i n t h e growth  - 44  of t h i s  -  o r g a n i s m m i g h t w e l l be t h e o x y g e n l e v e l i n t h e g r o w t h  menstruum.  Since 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 t h e s u r f a c e a r e a o f t h e a s s a y medium e x p o s e d  t o the  gas phase i s one o f t h e m a i n f a c t o r s d e t e r m i n i n g t h e oxygen a v a i l a b l e t o t h e c e l l  f o r growth.  To d e t e r m i n e t h e  TABLE V I . The  I n f l u e n c e o f S u r f a c e A r e a on t h e G r o w t h o f A. s u b o x y d a n s .  Amount o f C o m p l e t e b a s a l medium a d d e d t o each f l a s k (ml.)  in sq. cm.  5 10 15 20 Control ( n o t i n oculated)  influence  Surface Area  of t h i s  Growth Response m e a s u r e d as O p t i c a l Density Replicates 1 2  Average  18.103 16.989 16.626 15.385  0.940 0.959 0.796 0.552  0.921 0.940 0.810 0.569  0.930 0.949 0.803 0.560  16.989  0..027  0.022  0.025  f a c t o r &.n t h e g r o w t h r e s p o n s e  curve, the  t e s t o r g a n i s m was grown i n t h e b a s a l m e d i u n e n r i c h e d w i t h 0.05  micrograms  PABA i n 50 m l . E r y l e n m e y e r  flasks  v a r i o u s amounts o f medium p r e s e n t i n e a c h f l a s k . means t h e s u r f a c e a r e a o f t h e c u l t u r e s v a r i e d t o 1 8 . 1 0 3 s q . cm.  The r e s u l t s  from  response.  By t h i s 15.385  are given i n Table V I .  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 the growth  with  From  affect  The a r e a p r o v i d e d by t e n m l . o f  - 45  -  medium i n a 50 m l . f l a s k p e r m i t s o f maximum g r o w t h a t t h e 0.05  microgram  l e v e l o f PABA.  Such r e s u l t s w o u l d  suggest  t h a t t h e o x y g e n c o n t e n t o f t h e medium i s n o t l i m i t i n g i n t h e a s s a y p r o c e d u r e s i n c e 10 m l . o f medium a r e u s e d i n a 50 m l .  flask.  3•  Experiment  Influence  of I n c u b a t i o n Temperature  G r o w t h R e s p o n s e o f A. The A.  suboxydans.  s t e a d i n e s s of the i n c u b a t i o n temperature f o r  suboxydans  may  i n f l u e n c e t o a degree  of t h i s t e s t  o r g a n i s m , and t h u s may  irregularity  of the r e s u l t i n g  literature  on t h e  the growth  response  be t h e c a u s e o f t h e  assay curves.  i t i s n o t e d t h a t A. s u b o x y d a n s  From t h e  requires  an  i n c u b a t i o n t e m p e r a t u r e o f 30°C ( L a n d y 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 t h e 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 o f 2 8 - 3 5 ° C . conducted to observe the e f f e c t  E x p e r i m e n t s were  of i n c u b a t i o n  on t h e g r o w t h r e s p o n s e o f t h e t e s t  organism.  temperature Inoculated  a s s a y m e d i a were i n c u b a t e d a t 28~35°C, 30°C ( u s i n g a w a t e r bath set at t h a t temperature) and-27°C this  The r e s u l t s  experiment are presented i n Table V I I .  r e p r e s e n t a t i o n of these r e s u l t s  The  of  graphical  ( F i g u r e 8 ) shows t h a t  v e r y I r r e g u l a r g r o w t h r e s p o n s e c u r v e t o PABA by  a  sub-  o x y d a n s o c c u r r e d when t h e c u l t u r e s were i n c u b a t e d a t 28-35°C.  The  c u l t u r e s grown a t t h e s t e a d y t e m p e r a t u r e  30°C r e s u l t e d i n a l e s s  irregular  curve.  However t h e r e  of  TABLE V I I . I n f l u e n c e o f S t e a d i n e s s o f T e m p e r a t u r e on t h e G r o w t h R e s p o n s e s u b o x y d a n s t o PABA. Cone e n t r a t i o n o f P A B A / 1 0 cc B a s a l Medium i n micrograms 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1  I n c u b a t o r Temperature 28 - 3 5 ° C Average Replicates 1  2  0.114K 0.181 0.252 0.310 0.284 0.569 0.337 0.3.37 0.387 0.347 0.328  0.114 0.168 0.237 0.387 0.201 0.495 0.367 0.357 0.377 0.347 0.284  0.114 0.174 0.244 0.347 0.244 0.530 0.352 0.347 0 . 3 82 0.347 0.305  Water  Bath Temperature 30°C Average Replicates 1  2  0.114 0.131 0.237 0.292 0.201 0.237 0.337 0.337 0.377 0.328 0.276  0.114 0.161 0.227 0.310 0.208 0.260 0.328 0.357 0.301 0.292 0.328  K F i g u r e s r e p r e s e n t g r o w t h o f A. Optical Density.  0.114 0.146 0.234 0.296 0.205 0.256 0.333 0.347 0.337 0.310 0.301  suboxydans measured  as  o f A.  27°C  0.046 . 0.114 0.149 0.174 0.187 0.194 0.208 0.208 0.222 0.222 0.237  - 47 -  still test would  exists  a d e f i n i t e drop i n t h e growth  organism a t t h e 0.04 microgram have a n t i c i p a t e d t h e g r o w t h  level  response  of the  o f PABA.  o f A. s u b o x y d a n s  As one in 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. It  i s e v i d e n t t h a t a steady i n c u b a t i o n temperature i s de-  s i r a b l e but i t i s e q u a l l y evident that  temperature  a t i o n alone i s not s u f f i c i e n t . t o e x p l a i n  fluctu-  the v a r i a b i l i t i e s  of t h e response c u r v e .  E x p e r i m e n t 4s  The E f f e c t o f pH o f t h e Medium upon t h e G r o w t h o f A. s u b o x y d a n s  U n d e r k o f l e r e t a l . (1943) have s t u d i e d t h e i n f l u e n c e of t h e hydrogen response results  i o n c o n c e n t r a t i o n on t h e g r o w t h  o f A. s u b o x y d a n s  i n t h e a s s a y medium.  are presented i n Table  Their  VIII.  T h e i r r e s u l t s and t h o s e o f o t h e r s (Landy and D i c k e n , 1942j Cheldelin,  C h e l d e l i n a n d B e n n e t t , 1945; S a r e t t and  1945) s u g g e s t t h a t t h e optimum pH o f t h e c u l t u r e  medium a p p e a r s  t o be a p p r o x i m a t e l y s i x .  t h e p r e s e n t work were a d j u s t e d t o t h i s  The m e d i a u s e d i n  pH.  - 48  -  TABLE V I I I . E f f e c t of pH of t h e Medium upon t h e G r o w t h of _A. suboxydans"" ( U n d e r k o f l e r e_b al. 1943)  pH Before  of  G r o w t h M e a s u r e d as Optical Density  Medium  Sterilization After 5.30 6.10 6.90 7.10 7.75  Experiment  In L a n d y and culture  5:  48  hours  0.860 1.041 0.139 0.065 0.321  4.55 5.-95 6.75 7.18 7.78  hours  1.0'91 1.208 0.527 0.033 0.031  The E f f e c t o f Age o f I n i t i a l I n o c u l u m o f s u b o x y d a n s on t h e I r r e g u l a r i t y o f t h e Growth Response Assay Curve.  A.  the 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 PABA, Dicken  o f A.  other workers old  24  Sterilization  (1942)  u s e d a 24 h o u r  suboxydans (Sarett  and  o l d washed m o t h e r  f o r the i n i t i a l Cheldelin,  inoculum, while  1945) used a 3 0 - 4 0  hour  culture.  Hegarty  A c c o r d i n g t o Sherman and A l b u s ( 1 9 2 3 ,  1924)  (1939)  determines  a g r e a t degree ment.  For t h i s  desirable oculum  t h e p h y s i o l o g i c a l age the response  of c e l l s  of those c e l l s  r e a s o n i t was  t o determine whether  thought t h e age  that  response  curve.  to  t o a new  environ-  i t would  be  of the i n i t i a l i n -  d u r i n g t h e a s s a y p r o c e d u r e f o r PABA w o u l d  e f f e c t , on t h e g r o w t h  and  have  any  -  A series  49  -  o f a s s a y s were p r e p a r e d i n w h i c h t h e age  the i n i t i a l  inoculum d i f f e r e d .  A t w e n t y - f o u r h o u r and  forty-eight  hour mother c u l t u r e  o f A. s u b o x y d a n s  the i n o c u l a .  The age  of t h i s  b e a r i n g on t h e a d j u s t m e n t  p a r e n t c u l t u r e has  as r e p r e s e n t e d i n F i g u r e 9,  younger  of t h i s  experiment i n Table  whether  suboxydans  e x i s t and t h a t  PABA, i n t h e c u l t u r e  ir-  the  p a r e n t i n o c u l u m shows a g r e a t e r r e s p o n s e t o t h e  In the l i g h t  as  considerable  i t c a n be o b s e r v e d t h a t t h e  of t h e s e assay c u r v e s s t i l l  of t h e growth f a c t o r ,  level  medium.  of t h e above e x p e r i m e n t s t o d e t e r m i n e  a c e r t a i n f a c t o r or f a c t o r s would cause the i r -  regularity  of t h e a s s a y g r o w t h r e s p o n s e c u r v e of A.  oxydans , a p o s s i b l e e x p l a n a t i o n t h a t shows why all  were u s e d  However f r o m t h e g r o w t h r e s p o n s e c u r v e s o f A.  regularity  a  o f t h e o r g a n i s m s t o a f r e s h medium  as c a n be s e e n f r o m t h e r e s u l t s IX.  of  there e x i s t s  can now  be b r o u g h t  such pronounced  subforth  irregularity in  the assay experiments. These e r r a t i c  the "clumping e f f e c t " ment of t h e g r o w t h photometer,  g r o w t h r e s p o n s e c u r v e s may of the t e s t  organism.  be due  P r i o r t o measure-  o f t h e o r g a n i s m by t h e use o f t h e  spectro-  the c u l t u r e i s d i l u t e d t o double i t s volume  d i s t i l l e d water.  The  to  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  with so  t h a t t h e g r o w t h r e s p o n s e c a n be m e a s u r e d by means o f t h e resulting  turbidity  o f t h e c u l t u r e medium.  The  test  organism  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 o f t h e medium d u r i n g i t s growth  period.  Even a f t e r d i l u t i n g  t h e c u l t u r e and s h a k i n g  TABLE I X .  \  I n f l u e n c e o f Age o f P a r e n t I n o c u l u m o f A. s u b o x y d a n s on t h e G r o w t h Response t o v a r y i n g C o n c e n t r a t i o n o f PABA. Cone, o f PABA p e r 10 c c . o f B a s a l Medium i n Micrograms  24 h r . P a r e n t  x  Average  Replicates  0 .01 .02 .03 :o4 .05 .06 .07 .08 .09 .1 Figures  48 h r . P a r e n t I n o c u l u m  Inoculum  1  2  0.027* 0.268 0.319 0.398 0.398 0.301 0.252 0.602 0.658 0.602 0.569  0.032 0.284 0.337 0.409 0.284 0.292 0.244 0.552 0.602 0.585 0.620  Replicates  Average  1  2  0.022 0.181 0.276 0.337 0.284 0.319 0.337 0.456 0.409 0.357 0.367  0.027 0.168 0.252 0.337 0.284 0.328 0.337 0.495 0.409 0.337 0.357  r e p r e s e n t g r o w t h o f A. s u b o x y d a n s m e a s u r e d  as O p t i c a l  0.030 0.276 0.328 0.403 0.341 0.296 0.248 0.585 0.638 0.594 0.586  0.025 0.175 0.268 0.337 0.284 0.323 0.337 0.476 0.409 0.347 0.362 Density.  - 51  -  the r e s u l t i n g mixture t h o r o u g h l y , i t i s d i f f i c u l t  to  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 .  Instead,  of o b t a i n i n g  a single cell  suspension  c u l t u r e medium, t h e b a c t e r i a l c e l l s groups or clumps which the s i n g l e c e l l s . false  mittance  i s o b t a i n e d when s u c h  of a f l u i d  light-absorbing the presence material  suspension  i s influenced  cident  present  Quantitative  b a s e d upon t h i s  metry or nephelometry.  effect a  of t h e  test  not  the l i g h t  only  trans-  by t h e amount o f  i n s o l u t i o n but  also  light  by  (ex.  of m i c r o b i a l  p r i n c i p l e i s known as  These two  (ie. light  i n suspension,  analysis  although nephelometry  of s c a t t e r e d  of  i s measured t u r b i d i m e t r i c a l l y .  s u c h as i n s o l u b l e s u b s t a n c e s  equivalent,  form  o f 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  bacterial cells). in  clumping  (Hawk et a l . 1947)  material  to  homogeneous s u s p e n s i o n  of t h e growth response  In t u r b i d i m e t r y  the  have a tendency  As a r e s u l t o f t h i s  representation  organism  prevent  throughout  assure  cells  turbidi-  terms are s u b s t a n t i a l l y u s u a l l y r e f e r s t o the  at r i g h t angles  use  to the i n -  beam) as a measure of t u r b i d i t y . Turbidimetric  same p r o c e d u r e s substances  The  i n suspension  of the f l u i d  r e l a t i o n s h i p between t h e and t h e t u r b i d i t y o r  present  but  the of  amount  trans-  i s much more e m p i r i c a l t h a n f o r s u b -  s t a n c e s i n s o l u t i o n depending not material  c a r r i e d o u t by  i n s t r u m e n t s u s e d f o r t h e measurement  in solution.  of a s u b s t a n c e mittance  and  measurements c a n be  only  on t h e amount o f  a l s o on t h e s i z e and  shape o f  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  the  transparency,  - 52 -  t h e r e l a t i o n between light  particle  s i z e and t h e w a v e l e n g t h o f  used, and t h e u n i f o r m i t y w i t h w h i c h a g i v e n  turbidity  may be r e p r o d u c e d . I f a beam o f l i g h t contains p a r t i c l e s scattered.  i s p a s s e d t h r o u g h a medium t h a t  or b a c t e r i a l c e l l s ,  In c o l l o i d a l  as t h e T y n d a l l e f f e c t .  solutions this  the l i g h t w i l l  be  phenomenon i s known  I t isipossible to visualize  bacterial  cells  i n a homogeneous s u s p e n s i o n , and o f t h e same s i z e and  shape  having a r e l a t i v e  opacity or transparency.  a r e a b l e t o a b s o r b some l i g h t remainder.  and r e f l e c t  The r e f l e c t e d l i g h t From t h i s  bacterial cells  a r e n o t suspended  less  i t c a n be r e a d i l y as s i n g l e  lines  seen t h a t i f t h e cells  i n the  b u t r a t h e r as c l u m p s , t h e n e a c h c l u m p , b e i n g  l a r g e r and of d i f f e r e n t terial  cells  and t r a n s m i t t h e  i s d i r e c t e d on v a r i o u s  of r e f l e c t i o n .  solution,  These  cell,  shape  as c o m p a r e d t o a s i n g l e  a r e more opaque a n d a l s o w i l l  of t h e l i g h t  bac-  tend to r e f l e c t  beam.  As was m e n t i o n e d tenacious p e l l i c l e  p r e v i o u s l y , A. s u b o x y d a n s  on t h e s u r f a c e o f t h e medium.  forms a When t h e  c o n c e n t r a t i o n * o f PABA i n t h e a s s a y medium i s a t 0.03 t o 0.04 micrograms  p e r 10 c c , t h i s  a p p r o a c h a maximum.  pellicle  f o r m a t i o n appears t o  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 a t these concentrations.  The g r o w t h o f t h e o r g a n i s m has n o t  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 c o m p a r e d t o t h e l o w e r c o n c e n t r a t i o n o f PABA i n t h e medium, b u t t h a t t h e  - 53 -  formed  pellicle  on t h e s u r f a c e  when t h e d i l u t e d brings  about  the t e s t  a false  organism  Recently Surface that  culture  Active  prevents  i s shaken.  representation  a review  i n t h e form  they w i l l  not p r o d u c e  uniform  c l o u d i n g o f t h e medium.  throughout That t h i s  ester type  result  Furthermore,  and  but i n s t e a d a  than  as a s u r f a c e  of f a c t s .  diffusely pellicle.  o f Tween-  Growth d e v e l o p i n g i n  oleic  esters,  were e x t r e m e l y r e s i s t a n t  consisted of  to dispersion.  w i t h v e r y s m a l l amounts o f t h e w a t e r - s o l u b l e  ( 0 . 0 1 per c e n t Tween-80) growth was a t f i r s t  after  Tween-80  t o grow  was due t o t h e w e t t i n g p r o p e r t i e s  clumps which  will  i f b a c t e r i a are  (1946)-found t h a t  medium r a t h e r  t h e media c o n t a i n i n g n o n - w e t t i n g  ester  of a f i l m ;  noted  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  80 i s s u g g e s t e d by a number  large  I t was  by t h e medium, t h e y  mono-oleate) caused mycobacteria the l i q u i d  response of  on t h e a c t i o n o f  1949).  a pellicle  In 1946, Dubos and D a v i s  effect  of t u r b i d i t y .  of t h e l i t e r a t u r e  wetted,  of s o r b i t a n  of t h e g r o w t h  A g e n t s was done ( K i t t s ,  grow on the s u r f a c e  acid  This clumping  when measured by means  i f b a c t e r i a a r e not wetted  a fatty  homogeneous t u r b i d i t y  a few days  a fine  pellicle  formed  submerged  a t the s u r f a c e  of t h e medium, p r o b a b l y due t o t h e e x h a u s t i o n of t h e s u r f a c e activator With  by h y d r o l y s i s  further  wetting  addition  of t h e c e l l s ,  throughout  and u t i l i z a t i o n  by t h e o r g a n i s m s .  o f Tween-80 t o t h e medium, c a u s i n g the microbial  t h e medium.  population dispersed  - 54 -  Woods ( 1 9 4 9 ) 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 t h e growth  o f A. s u b o x y d a n s .  adding t h i s w e t t i n g agent  He f o u n d t h a t by  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 o f 0.5 p e r c e n t t h e f o r m a t i o n o f t h e p e l l i c l e on t h e s u r f a c e o f t h e medium was p r e v e n t e d . developed throughout  the l i q u i d  The  organism  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 it  may be p o s s i b l e t o overcome t h e i r r e g u l a r i t y  response  workers,  o f t h e growth  c u r v e o f A. s u b o x y d a n s t o i n c r e a s i n g i n c r e m e n t s o f  PABA by a d d i n g Tween-80 t o t h e c u l t u r e medium b e f o r e i n o c u lation.  Experiment  6r  The Use o f Tween-80 t o Submerge t h e G r o w t h of  A. s u b o x y d a n s d u r i n g t h e A s s a y f o r PABA.  A s e r i e s o f 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 o f PABA u s i n g t h e method o f L a n d y a n d D i c k e n but adding d i f f e r e n t concentrations  c o n c e n t r a t i o n s o f Tween-80.  of t h i s  surface activator  (1942), The  final  i n the growth  medium  were 0.1, 0.25, a n d 0.5 p e r 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 w e r e u s e d t o d e t e r m i n e  what l e v e l was  to  prevent the 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  suitable  organism.  F i g u r e "10 r e p r e s e n t s t h e r e s u l t s t a b u l a t e d i n T a b l e X.  It  c a n be s e e n t h a t Tween-80 a t a l e v e l o f 0.5 p e r c e n t i n t h e c u l t u r e medium p r e v e n t s t h e f o r m a t i o n o f a p e l l i c l e A. s u b o x y d a n s , test  and t h u s t h e growth  response  o r g a n i s m t o PABA t e n d s t o y i e l d  by  curve of t h i s  a smooth c u r v e .  TABLE X. The I n f l u e n c e o f Tween-80 on t h e G r o w t h R e s p o n s e o f A. s u b o x y d a n s t o I n c r e a s i n g I n c r e m e n t s o f PABA.  Cone, o f PABA i n micrograms p e r 10 c c . b a s a l medium 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1  No Tween 0 . 1 p e r c e n t Tween80 0.018H 0.143 0.237 0.306 0.301 0.392 0.420 0.523 0.516 0.488 0.482 x  0.25  p e r c e n t Tween- 0 . 5 p e r c e n t Tween80 80 0.013 0.140 0.237 0.268 0.314 0.347 0.495 0.523 0.509 0.523 0.500  0.013 0.149 0.252 0.310 0.357 0.398 0.444 0.482 0.516 0.509 0.538  F i g u r e s r e p r e s e n t g r o w t h o f A. s u b o x y d a n s m e a s u r e d as Optical Density,  0.013 0.102 0.284 0.319 0.367 0.398 0.482 0.469 0.482 0.658 0.670  - 56 -  The  other tested l e v e l s  o f Tween-80 i n t h e a s s a y medium, 0.1  and  0.25 p e r c e n t , w e r e n o t a d e q u a t e t o p r e v e n t  this  surface  film formation. Additional series u s i n g t h e same p r o c e d u r e  o f a s s a y d e t e r m i n a t i o n s f o r PABA and i n c o r p o r a t i n g t h e s u r f a c e  a c t i v e agent  were done t o c h e c k  t h e above r e s u l t s .  these checks  (Table X I ) t h e growth  response  drawn t o show t h a t t h e p r e v i o u s r e s u l t s  From  curves are  c a n be  reproduced  and f u r t h e r t h a t t h e a s s a y s  h a v i n g no Tween-80 c o n t i n u e t o  p r o d u c e an i r r e g u l a r  response  III.  growth  curve  (Figure 11).  B i o l o g i c a l Aspects i n R e l a t i o n t o Assay Procedure: G r o w t h R e q u i r e m e n t s f o r A. s u b o x y d a n s . In a d d i t i o n t o t h e establishment o f the assay  cedure  f o r t h e ' d e t e r m i n a t i o n o f PABA i t i s f e l t  d i s c u s s i o n of t h e growth  requirements  s h o u l d be i n c l u d e d a t t h i s  point.  these biochemical aspects w i l l  that a  of t h i s test  For purposes  pro-  organism  of c l a r i t y  be d i s c u s s e d u n d e r  separate  headings. (a) Carbon The  Source:  carbon  source that i s i n c o r p o r a t e d i n t o  t h e c u l t u r e medium f o r A. s u b o x y d a n s i s g l y c e r o l . organism to  secures growth  dihydroxyacetone:  energy  by t h e o x i d a t i o n  This  of g l y c e r o l  TABLE X I . The I n f l u e n c e o f Tween-80 on t h e G r o w t h R e s p o n s e o f A. s u b o x y d a n s t o I n c r e a s i n g I n c r e m e n t s o f PABA. Cone, o f PABA  Added Tween-80 ( . 5  p e r 10 c c , b a s a l medium 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1  Replicates 1 2 0,004* 0.015 0.066 0.119 0.210 0.328 0.392 0.444 0.482 0.523 0.550  *  0.004 •0.015 0.051 0.108 0.206 0.337 0.401 0.452 0.495 .0.513 0.522  No Added Tween-80  percent) A  Replicates B  0.013 0.218 0.367 0.469 0.530 0.568 0.595 0.620 0.648 0.658 O.658  0.011 0.230 0.387 0.495 0.545 0.576 0.602 0.627 0.640 0.655 0.658  1 0.000 0.004 0.009 0.036 0.056 0.071 0.114 0.237 0.276 0.284 0.284  F i g u r e s r e p r e s e n t g r o w t h o f A. s u b o x y d a n s m e a s u r e d i n t e r m s of O p t i c a l D e n s i t y .  A 0.018 0.182 0.347 . 0.398 0.495 0.523 0.530 0.538 0.658 0.648 0.638  -  58  -  CH 0H  CH 0H  2  2  CHOH  *  C=0  CH 0H 2  In recent  studies  i t was  products a f t e r being k.  a c t i o n on  noted that glucose  a u t o c l a v e d ) had  s u b o x y d a n s when t h i s  carbon source the is altered quite  character  carbohydrate  source  i s u s e d as  g r o w t h of t h e  organism  Source;  S t o k e s and mining f o r the f i r s t  Larsen  time the  (1945) succeeded i n  specific nitrogen  These i n v e s t i g a t o r s n o t e d t h a t  t w e n t y amino a c i d s  s t u d i e d v a l i n e was  i n i t s absence.  probably  essential,  as shown i n T a b l e X I I . absolute  s i n c e the  a b s e n c e may as an  be  due  other  s t i m u l a t o r y and  no were  non-essential  n e e d f o r i s o l e u c i n e may  s y n t h e t i c l e u c i n e of the  be in i t s present  medium  1944).  I t i s i n t e r e s t i n g t o note t h a t the w i t h the  the  amino a c i d s  t o a s m a l l amount o f i s o l e u c i n e  Wardell,  of  essential since  s m a l l amount o f g r o w t h o b t a i n e d  i m p u r i t y i n the  ( H e g s t e d and  The  The  deter-  requirements  o f A,, s u b o x y d a n s .  c l a s s e d as  a  significantly.  (b) N i t r o g e n  growth occurred  was  Cheldelin, 1945).  t h a t when g l u c o s e of the  end-  a marked s t i m u l a t o r y -  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 However, Woods ( 1 9 4 9 ) c l a i m s  (or i t s  t w e n t y amino a c i d s i n c o r p o r a t e d  medium was  about e q u a l  to that  c a s e i n was  used i n t h e i r  place  obtained (Table  growth  i n the when  XIII).  obtained  culture  hydrolyzed  -  59  -  TABLE X I I . E f f e c t o f O m i s s i o n o f I n d i v i d u a l Amino A c i d s f r o m t h e " 2 0 amino a c i d medium" upon G r o w t h o f A. subxixydans ( S t o 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  Essential  Stimulatory  Non  -68  Histidine-45  Leucine  Isoleucine  Valine  -98H  Alanine  -72  Cystine Glutamic acid Aspartic acid Proline Hydroxyproline  -37 -41 -38 -39 -38  Essential -31  Methionine -32 T r y p t o p h a n e -30 Tyrosine  -33  Phenylalanine -28 Threonine -30 Lysine -33 Arginine -32 Serine -28 N o r l e u c i n e -29 Glycine -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 o f c u l t u r e s grown w i t h o u t t h e 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 o f 3 2 . TABLE  XIII  G r o w t h o f A. s u b o x y d a n s w i t h V a r i o u s C o m b i n a t i o n s o f Amino Ac i d s . ( S t o k e s and L a r s e n . 1945) Amino A c i d s i n B a s a l  Medium  Valine 4 isoleucine + alanine + 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 Hydrolyzed casein K  Per cent T r a n s m i s s i b l e L i g h t E x p e r i m e n t A E x p e r i m e n t B. 52x  50  29 38 32 41 29 17  34 37 40 37 29 27  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 .  -  S t o k e s and  60  Larsen  -  (1945) were a b l e  t o show t h a t  c o m b i n a t i o n o f v a l i n e , i s o l e u c i n e , a l a n i n e and represented  the  by t h e  histidine  s m a l l e s t number o f amino a c i d s w h i c h  c o n s i s t e n t l y s u p p o r t some g r o w t h o f A,  a  suboxydans.  can However  a d d i t i o n of e i t h e r c y s t i n e o r m e t h i o n i n e g r o w t h  considerably  improved.  growth again  was  mixture  of the  W i t h the f u r t h e r a d d i t i o n of p r o l i n e ,  increased  to the  t w e n t y amino a c i d s  However d e v e l o p m e n t i s n o t s i v e t h a n w i t h t h e use  so  level  t r e a t e d p e p t o n e and  or h y d r o l y z e d  r a p i d and  of y e a s t  Norite A-treated  growth r e s p o n s e of t h i s  organism.  (c) Mineral  variation,  t h e y must c o n t a i n t e n known  elements:  exact  obtained  a  to  greater  i t i s believed  microthat  s a l t s are necessary f o r b a c t e r i a l n u t r i t i o n  inorganic  vary with  u s u a l l y designated  S o l u t i o n A and  S o l u t i o n B.  c e n t K H P 0 , and  10 p e r  each  i n two  groups,  2  This  A.  namely  S o l u t i o n A c o n s i s t s o f 10  cent K HP0^.  and  species.  s a l t s a d d e d t o t h e b a s a l medium o f  suboxydans are  2  concentrate  (6) calcium (7) p o t a s s i u m ( 8 ) magnesium (9) sulphur (10) phosphorus  carbon " hydrogen oxygen iron nitrogen  requirements probably  The  A-  f o r m s o f f o o d r e q u i r e d by  o r g a n i s m s show c o n s i d e r a b l e  the  exten-  Source;  Although the  inorganic  the  casein.  somewhat l e s s  liver  s u b o x y d a n s and  The  with  extract.  t h e b a s a l medium f o r A.  (1) (2) (3) (4) (5)  obtainable  C h e l d e l i n (1945) i n c l u d e d N o r i t e  S a r e t t and  was  per  solution i s usually  -  61 -  added t o t h e medium b e c a u s e i t p o s s e s s e s a s t r o n g b u f f e r i n g S o l u t i o n B c o n s i s t s of 4 per cent  action. 0.2  per cent  MnS0^.4H 0. 2  for  order  0.2 per cent  A l l these  the v i t a l  protoplasm. in  NaCl,  salts  MgS0^.7H 0, 2  F e S 0 ^ . 7 H 0 a n d 0.2 p e r c e n t 2  provide  the necessary  f u n c t i o n s and a c t i v i t i e s  elements  of the b a c t e r i a l  The N a C l i s g e n e r a l l y a d d e d t o c u l t u r e m e d i a  to increase  their  osmotic pressure.  medium i s one t h a t c o n t a i n s  riot  A  satisfactory  only the necessary  food  s u b s t a n c e s i n p r o p e r p r o p o r t i o n s b u t a l s o an o s m o t i c approaching i s o t o n i c i t y with the p a r t i c u l a r During  cell.  the p r e l i m i n a r y assay determination  u s i n g t h e method of Landy and D i c k e n ( 1 9 4 2 ) , t h a t t h e b a s a l medium f o r t h e t e s t prepared  appeared opalescent  must be due t o t h e f o r m a t i o n reaction carried order this  being  cloudiness  o f a p r e c i p i t a t e by a  o f two o r more compounds.  chemical  A s e r i e s o f t e s t s were  o u t on a l l t h e c o n s t i t u e n t s o f t h e b a s a l medium i n  t o f i n d what c o n s t i t u e n t o r c o n s t i t u e n t s p r o d u c e d precipitation.  presented the  This  o f PABA  i t was n o t e d  organism a f t e r  or cloudy.  pressure  The r e s u l t s  i n Table XIV.  cloudiness  of t h i s  experiment are  I t c a n be s e e n f r o m t h i s t a b l e  o f t h e b a s a l medium a r i s e s on  S o l u t i o n s A and B. turbidimetrically,  that  mixing  S i n c e t h e growth r e s p o n s e i s measured t h e b a s a l medium must be as c l e a r a s  p o s s i b l e , t h e r e f o r e i t was f o u n d d e s i r a b l e t o f i l t e r t h e medium t h r o u g h a S e i t z f i l t e r i n g avoid formation  pad ( t y p e ST, s i z e L 6 ) t o  of a p r e c i p i t a t e during  autoclaving.  - 62 TABLE I V . The P e t e r m i n a t i o n of,, t h e C a u s a t i v e F a c t o r s t h a t P r o d u c e O p a l e s c e n c e i n t h e B a s a l Medium.  Constituent  Sol.  A - ( K  Sol.  (KH P0, B-MgS0..7H 0 NaCl  2  Character of S o l u t i o n Before Autoclaving  Character of Solution After Autoclaving  clear  clear  H P 0 A 2  2  clear  FeS04.7H 0 ?  Sol.  MnSO,.4H,0 A + S o l . B^  Tryptophane s o l n . Casein Hydrolysate Nicotinic Acid soln. Cystine solution Ca. Pantothenate Tryptophane4Sol. A  Tryptophane+Sol. B Tryptophane-Sol. A and S o l . B Casein Hydrol. + Sol. A Casein Hydrol. + Sol. B Casein Hydrol. + S o l . A and S o l . B  cloudy, white ppte. clear clear cloudy, white ppte. cloudy, white ppte. cloudy, white ppte.  Cystine Sol. 4 S o l . A and S o l . B Nicotinic acid S o l . + Sol. B + Sol. B  (d) V i t a m i n As requirements found  white cloudy precipitate clear clear clear clear clear clear clear  s l i g h t pale y e l l o w ppte heavy w h i t e ppte clear clear clear clear clear clear s l i g h t pale y e l l o w ppte. heavy white ppte. s l i g h t white ppte. s l i g h t pale y e l l o w ppte. heavy white ppte. heavy white ppte. heavy w h i t e ppte.  Source;  a r e s u l t of t h e i r  s t u d y on t h e g r o w t h  o f JU s u b o x y d a n s , U n d e r k o f l e r e t a l . ( 1 9 4 2 )  that besides  a s u i t a b l e .carbon  n i t r o g e n and m i n e r a l s a l t s ,  source,  organic  t h e medium must c o n t a i n  -  63 -  pantothenic acid, n i c o t i n i c would  occur.  acid  and PABA, b e f o r e g r o w t h  I n a d d i t i o n i t was c o n c l u d e d t h a t  riboflavin  was n o t r e q u i r e d i n t h e medium; i t a p p e a r e d t h a t A,, s u b o x y dans had t h e 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  w o r k e r s 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. 1 0 0 0 a n d 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 not  factor,  b i o t i n , w h i c h was i n d i s p e n s a b l e f o r t h e g r o w t h o f t h e  organism.  This e s s e n t i a l  investigators  factor  t o be n i c o t i n i c  has b e e n f o u n d by o t h e r  acid.  (e ) P u r i n e M e t a b o l i s m ' ; L a n d y and S t r e i g h t o f f a variety  (1943)  o f compounds e x a m i n e d f o r a b i l i t y  g r o w t h o f A,, s u b o x y d a n s  observed that of to increase  at low concentrations  o f PABA i n t h e  b a s a l medium, t h e p u r i n e b a s e s , a d e n i n e , q u a n i n e , and x a n t h i n e were f o u n d 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 o f g r o w t h i n c r e a s e d , b u t a l s o t h e s e n s i t i v i t y o f A. suboxydans absence  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 .  While i n the  of purines the t e s t organism responds t o 0.01 m i c r o -  grams o f PABA, t h e p r e s e n c e o f p u r i n e s b r i n g s a b o u t measurable  growth w i t h 0 . 0 0 1 micrograms  above i n v e s t i g a t o r s  per c u l t u r e .  are  s u g g e s t i o n because  without effect  The  f o u n d t h a t t h e s e p u r i n e s were n o t e s -  s e n t i a l f o r g r o w t h b u t as g r o w t h a c c e s s o r i e s . forth this  readily  They b r o u g h t  they noted that the purines  i n t h e absence  o f PABA o r when PABA was  s u p p l i e d i n amounts above 0 . 0 3 m i c r o g r a m s  per 10 m l . o f  culture. T h e r e i s s t r o n g e v i d e n c e t h a t PABA i s c o n c e r n e d i n  - 64 the  synthesis  methionine  o f p u r i n e s , as w e l l as f o l i c  (Woods, 1947).  Since this  a c i d , t h y m i n e and  i s t h e c a s e i t may  suggest t h e reason 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. of  These compounds o r p o s s i b l y one  them, may be s y n t h e s i z e d b y A., s u b o x y d a n s  factor,  PABA.  from t h e growth  Hence when t h e p u r i n e s a r e a d d e d t o t h e b a s a l  medium i n w h i c h t h e r e i s a l o w c o n c e n t r a t i o n o f PABA, t h a t w o u l d 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 readily accessible  purines permit normal  of  The s m a l l amount o f PABA i n t h e medium  A. s u b o x y d a n s .  w o u l d t h e n be u s e d by t h i s other v i t a l  IV.  Correlation  organism i n t h e s y n t h e s i s of  of T u r b i d i t y Readings w i t h A c t u a l Counts.  discussed i n this  series  occasionally  o r g a n i s m h a s been o b t a i n e d i n t h e s y n t h e t i c  medium t o w h i c h no PABA has b e e n a d d e d . u s e d as t h e i n i t i a l  inoculum i n the assay  well contain sufficient initiation  Cell  o f a s s a y s u n d e r t a k e n and t h o s e  r e p o r t , i t c a n be n o t e d t h a t  growth of t h e t e s t  In  metabolism  factors.  From t h e l a r g e  the  cellular  growth,  o f growth  t h e work o f L a n d y  The washed  cells  procedure-might  PABA o r a c o n j u g a t e o f PABA t o p e r m i t i n a medium d e v o i d o f added PABA.  and D i c k e n (1942) a n d o t h e r w o r k e r s  (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 a t t e m p t was made t o d e t e r m i n e the  number o f c e l l s  biological  present i n the inoculum f o r the micro-  assay f l a s k s .  Therefore f o r t h i s reason i t  - 65 -  seemed d e s i r a b l e t o d e t e r m i n e t h e number o f b a c t e r i a l u s e d as i n i t i a l and M i s r a  inoculum.  cells  The d r o p p l a t e t e c h n i q u e o f M i l e s  (1938) as d e s c r i b e d i n A p p e n d i x V was u s e d t o  determine the b a c t e r i a l count. glycerol-yeast  The o r g a n i s m was p l a t e d on  e x t r a c t agar (Appendix I V ) .  Data were ob-  t a i n e d f r o m 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 a r e p r e s e n t e d in  T a b l e XV. TABLE XV. P l a t e C o u n t s o f Number o f C e l l s p e r m l . of Washed M o t h e r C u l t u r e .  Determination Number  Log. of Count/ml of M o t h e r Culture  9.20 8.57 8.46  1 2 3  From t h i s of  "size"  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  I t w o u l d be o f i n t e r e s t t o t e s t t h e i n f l u e n c e o f  of i n i t i a l  i n o c u l u m on t h e g r o w t h r e s p o n s e 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 ml.  7.90 7.25 7.15  d a t a i t c a n be e s t i m a t e d t h a t t h e number o f c e l l s  A. s u b o x y d a n s  2.5 x 10^.  Calculated Log. Count o f I n o c u l u m used i n Assay Flasks  A series  micrograms  o f PABA p e r 10  o f 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  f r o m a washed m o t h e r c u l t u r e o f known c e l l  dilutions  concentration.  T a b l e X V I p r e s e n t s t h e r e s u l t s o f two s u c h d e t e r m i n a t i o n , the  d a t a f r o m No. 2 b e l o w ,  are p l o t t e d  i n Figure  12.  -  66 -  TABLE X V I . The I n f l u e n c e o f I n i t i a l I n o c u l u m C o u n t on t h e R e s p o n s e o f A. s u b o x y d a n s when Grown 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 m i c r o g r a m s o f PABA p e r 10 m l . o f Medium.  No. 1 C e l l Count p e r 0.05 c c . of Inoculum  No. 2 T u r b i d i t y of C u l t u r e a t 72 h r s . , measured as O p t i c a l Density  B l a n k No PABA , 0 . 5 x lOr 0.25 x 1CK 0.5 x 1 0 1 . 0 x 103 2.0 x 1 0 4.0 x 1 0 4 . 0 x 104 8.0 x 10' 3  3 3  It  numbers o f c e l l s ,  3 3  3  6  response  i n the i n i t i a l  as t h a t  i s proportional to  inoculum.  Equivalent  o f No. 2 , a b o v e , w e r e i n o c u l a t e d  i n t o t h e b a s a l medium d e v o i d o f PABA. measureable  0.000 0.002 0.009 0.052: 0.108 0.588 1.086 1.188  3  i s e v i d e n t t h a t t h e growth  t h e number o f c e l l s  T u r b i d i t y of C u l t u r e a t 48 h r s . , measured as O p t i c a l Density  B l a n k No PABA. .0.058 x 1 0 x 10 0.29 x 10 0.58 x 10 2.9 x loj 2.9 x. 10? 2.9 x 10 2.9 x 10? 2.9  0.003 0.068 0.180 0.279 0.751 0.770 0.842 0.947  5  C e l l Count p e r 0.1 c c . of Inoculum  amount o f g r o w t h  In every f l a s k  resulted.  In t h i s  no  particular  c a s e t h e r e i s no e v i d e n c e t o s u g g e s t t h a t w i t h h e a v y  inocula  t h e r e i s a n y c a r r y - o v e r o f PABA o r 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.  Consequently  i f the parent c u l t u r e i s adequately  washed w i t h s a l i n e b e f o r e b e i n g u s e d t h e r e seems t o be l i t t l e assay  flasks.  as t h e i n i t i a l  inoculum,  o r no c a r r y - o v e r o f PABA i n t o t h e  EfYCE  B  0  O  05  -  67  -  These e x p e r i m e n t s have i n d i c a t e d , cell the at  c o n c e n t r a t i o n i n the i n i t i a l subsequent  r a t e of g r o w t h .  inoculum markedly  I t might  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  cell  however, t h a t  be w i s e t o s u g g e s t  connection, that  If this  the  c o u l d be done, u s i n g t h e same p r o c e d u r e  technique i n p r e p a r i n g the parent c u l t u r e , the response  o f A.  and  a standard curve  suboxydans t o v a r y i n g i n c r e m e n t s  of  T h i s ' c u r v e c o u l d t h u s be u s e d  as a  PABA c o u l d be o b t a i n e d .  means o f c a l c u l a t i n g l e v e l s this  affects  c o n c e n t r a t i o n o f 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  level.  for  the  o f PABA by g r o w t h r e s p o n s e  of  t e s t organism, without p r e p a r i n g a r e f e r e n c e assay  each t i m e a d e t e r m i n a t i o n o f t h i s  compound i s s o u g h t .  TABLE X V I I . R e l a t i o n s h i p Between C e l l Cell  In  0.504 0.284 0.248 0.212 0.161 0.096 0.056  s  connection Table XVII presents the  s h i p between c e l l 48 h o u r  O p a c i t y , m e a s u r e d as Optical Density  x 10° x 10° x 10° x 10° x 10° x 10* x 10  this  and  Suspension Opacity.  Number o f C e l l s per c c . 2.90 1.45 1.27 1.07 0.84 0.58 0.29  Count  c o u n t and c e l l  o l d c u l t u r e o f A.  relation-  suspension opacity using a  suboxydans.  As was  anticipated  the  i n c r e a s e i n o p a c i t y of t h e 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 o f c e l l s  (Figure  13).  - 68 -  V.  F i n a l Recommended A s s a y From t h e r e s u l t s  Procedure:  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 ,  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 PABA c a n t h u s recommended. of  T h i s a s s a y p r o c e d u r e has b e e n a d o p t e d  the experiments  t o f o l l o w and i t i s f e l t  be  f o r many  t h a t such  a  p r o c e d u r e s h o u l d be u s e d i n f u t u r e work a l o n g t h e l i n e s the  proposed  Dicken (1942)  a s s a y p r o c e d u r e o u t l i n e d by L a n d y  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 ,  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 p u r e .  medium d e s c r i b e d by t h e s e w o r k e r s for  of  experimentations reported herein. The  all  an  the t e s t  appears  t o be  being sure The  o f A.  amounts o f PABA i s r e q u i r e d ,  medium d e s c r i b e d by L a n d y and S t r e i g h t o f f  (1943)  (1945)  of  organism i s measured t u r b i d i m e t r i c a l l y ,  i s recommended.  medium b e f o r e s t e r i l i z a t i o n  and  Cheldelin  S i n c e the growth  a d d i t i o n of the S u r f a c e A c t i v e Agent,  response the  Tween-80, t o t h e  subsequent  inoculation,  recommended i n o r d e r t o p r e v e n t t h e f o r m a t i o n o f a  sub-  the  or  and B e n n e t t the t e s t  bas.al  satisfactory  o r g a n i s m , however i f g r e a t e r r e s p o n s e  oxydans t o v e r y minute  and  basal is  pellicle  on t h e s u r f a c e o f t h e medium.  VI.  The  Biological  A c t i o n of U l t r a v i o l e t  Irradiation:  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 b a s e d on t h e 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 o f t h e "essential  m e t a b o l i t e " PABA, may  an e x t e n t t h a t t h e new  a l t e r t h a t compound t o  compound f o r m e d  such  is sufficiently like  -  its  non-irradiated  ation This in  69 -  precursor  in a cellular  that  system i n p l a c e  i t blocks  normal m e t a b o l i c lite"  hibition  function concerning  or death  of the c e l l  subsequent a  synthetic  Experiment  t o permit  7:  e x p e r i m e n t s were c a r r i e d o u t irradiation  t h e growth  o f PABA on i t s  o f _A. s u b o x y d a n s i n  E f f e c t of U l t r a v i o l e t Light V a l u e o f PABA.  M e r c u r y A r c Lamp  to the b a s a l  p e r 10 m l .  the General  Electric  ( s e e A p p e n d i x V I ) f o r 30, 45, 60  The i r r a d i a t e d PABA s o l u t i o n was t h e n  medium a t a l e v e l After  medium was t h e n  on t h e B i o l o g i c a l ; •  2 micrograms  w a t e r was i r r a d i a t e d under  90 m i n u t e s .  o f medium.  of i n -  medium.  of d i s t i l l e d  and  so t h a t t h e  t h e " e s s e n t i a l metabo-  A s o l u t i o n o f PABA c o n t a i n i n g  Germicidal  anti-metabolite  i n question.  of u l t r a v i o l e t  ability  combin-  metabolite.  r e s u l t may be a s t a t e  A s e r i e s of p r e l i m i n a r y the influence  an  an e s s e n t i a l enzyme s y s t e m  i s stopped, the f i n a l  into  of the normal  n e w l y f o r m e d compound may a c t l i k e  that  on  i t may e n t e r  sterilization  inoculated  washed mother c u l t u r e  with  o f 0.05 m i c r o g r a m s and s u b s e q u e n t  p e r 10 m l .  c o o l i n g the  0.05 m l . o f a 48 h o u r o l d  o f A. s u b o x y d a n s .  experiments are presented  added  The r e s u l t s o f t h e s e  i n T a b l e s X V I I I and X I X .  R e f e r e n c e t o t h e above d a t a  i n d i c a t e s that  radiation  o f PABA u n d e r t h e c o n d i t i o n s  a partial  destruction  described,  the i r results i n  o f t h e 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 o f I r r a d i a t i o n o f PABA on i t s Subs e q u e n t Use a s a n E s s e n t i a l M e t a b o l i t e f o r A. s u b o x y d a n s . F l a s k No. Replicates  1 2 3 4 5 6 7 8  No PABA  0.068* 0.068 0.068  --  — —  --  0.068  Average  Normal PABA Enrichment  30-Minute Irradiated PABA  45-Minute Irradiated PABA  0.961 0.959 0.959 0.959 0.959 0.921 Q.943 0.930  0.618 0.620 0.620 0.638 0,654 0.654 0.654 0.660  0.328 0.367 0.347 0.367 0.367 0.301 0.347 0.076  0.949  0.639  0.341  TABLE X I X . The E f f e c t o f I r r a d i a t i o n o f PABA on i t s Subs e q u e n t 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. s u b o x y d a n s .  F l a s k No. Replicates  No PABA  1 2 3 4 5 6 7 8  0.029K 0.027 0.029 —  Average  0.028  -— — —  x Figures  Normal PABA Enrichment  60-Minute Irradiated PABA  90-Minutee Irradiated PABA  0.783 0..783 0.783 0.839 0.839 0.783 0.839 0.796  0.071 0.053 0.056 0.052 0.051 0.076 0.076 0.076  0.036 0.034 0.032 0.029 0.041 0.043 0.029 0.031  0.806  0.064  0.034  r e p r e s e n t g r o w t h o f A. s u b o x y d a n s measured as O p t i c a l D e n s i t y .  - 71 -  an " e s s e n t i a l m e t a b o l i t e " i s c o n c e r n e d . o b t a i n e d a f t e r 45 m i n u t e s  truction partial  since  sufficient  a p p r e c i a b l e growth  The e x t e n t o f d e s -  of i r r a d i a t i o n  u n a l t e r e d PABA r e m a i n e d  i s only t o permit  o f t h e o r g a n i s m a l t h o u g h t h e g r o w t h was  not n e a r l y equal 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 A f t e r 90 m i n u t e no g r o w t h  irradiation  of the t e s t  of a s o l u t i o n  PABA.  o f PABA l i t t l e t o  o r g a n i s m o c c u r r e d , w h i c h c o u l d be due  t o t h e f a c t t h a t a l l b u t a s m a l l amount o f t h e 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 t h e p l a c e o f n o r m a l 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  Experiment The  8:  T i m e - D e s t r u c t i o n Curve  o f PABA.  above e x p e r i m e n t was r e p e a t e d u s i n g a s e r i e s o f  irradiation times. in  metabolite".  The d a t a f r o m t h i s  T a b l e s XX a n d X X I .  experiment a r e presented  The r e s u l t s t a b u l a t e d i n T a b l e XX a r e  p l o t t e d i n F i g u r e 14 and 1 5 . Irradiation  of a 2 microgram  solution  o f PABA f o r 3 0 m i n u t e s  truction  o f t h e PABA; i r r a d i a t i o n  86 p e r c e n t d e s t r u c t i o n . similar tions  p e r 10 m l . aqueous  r e s u l t e d i n 50 p e r c e n t  des-  f o r 60 m i n u t e s r e s u l t e d i n  In the l i g h t  of these r e s u l t s a  e x p e r i m e n t was c o n d u c t e d i n w h i c h v a r i o u s c o n c e n t r a -  o f PABA were i r r a d i a t e d , by u l t r a v i o l e t  light.  E x p e r i m e n t 9 :• T i m e - D e s t r u c t i o n o f V a r i o u s C o n c e n t r a t i o n s o f PABA b y 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  o f PABA, 1 0 0 , 5 0 , 10 and 2  TABLE XX. Time o f I r r a d i a t i o n  Replicate F l a s k No.  No PABA  - Destruction  Normal PABA  Time 15  1 2 3 4 5 Average  Data f o r I r r a d i a t e d  30  45  of 60  PABA.  Irradiation 70  80  0.014K 0.014 0.014 — --  0.854 0.806 0.798 0.824 0.858  0.796 0.796 0.783 0.819 0.796  0.494 0.509 0.485 0.329 0.454  0,324 0.276 0.315 0.237 O.365 0.284 0.346 0.286 0.339 0,284  0.137 0.137 0.117 0.121 0.168  0.014  0.830  0.799  0.476  0.336  0 . 1 3 5 O.O85  K Figures represent  0.273  0.076 0.094 O.O89 0.083 O.O83  in 90  Minutes 100  110  0.076 0.068 O.O63 0.071 0.061  0.071 0.077 0.028 0.030 0.032 0.011 0.018 0 . 0 0 0 0.004 0 . 0 0 0  0.068  0.029  growth o f A. s u b o x y d a n s measured as O p t i c a l  0.022  Density.  TABLE X X I . Time o f I r r a d i a t i o n  Replicate F l a s k No.  1 2 3  Average  No PABA  O.OOC-x  0.000 0.000 0.000  Normal PABA  0.523 0.523 0.523 0.523  - 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  15 0.469 0.301 0.482 0.409  x ' F i g u r e s r e p r e s e n t g r owth  Time  of  30  45  0.155 0.131 0.181  0.155  0.056  0.018  0.051 0.046  Irradiation  60 0.041 0.009 0.027 0.027  in  80  0.004 0.013 0.009 0.009  o f A. s u b o x y d a n s m e a s u r e d  PABA.  Minutes  100  120  140  0.004 0.004  0.004 0.000 0.000 0.000  0.004 0.000 0.000 0.000  0.004 0.004  as O p t i c a l  Density.  - 74 -  m i c r o g r a m s p e r 10 m l . d i s t i l l e d w a t e r , 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  added t o a b a s a l c u l t u r e medium a t t h e l e v e l grams p e r 10 m l . o f medium. w i t h 0.05 dans . the  The c u l t u r e s w e r e s u b s e q u e n t l y  inoculated  incubated  a t 30°C a n d  a t t h e e n d o f a 48 a n d a 72  The r e s u l t s a r e p r e s e n t e d  i n Table  F i g u r e 16 g i v e s t h e g r a p h i c a l r e p r e s e n t a t i o n o f t h e  results  obtained. From t h e 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  determine the r e a c t i o n order  alteration  of the d e s t r u c t i o n or  o f PABA b y u l t r a v i o l e t  light.  From t h e 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 o f r e a c t i o n r a t e s , are  micro-  m l . o f a 24 h o u r washed m o t h e r c u l t u r e o f A. s u b o x y -  hour i n c u b a t i o n p e r i o d .  to  o f 0.05  These m e d i a were t h e n  g r o w t h r e s p o n s e was r e c o r d e d  XXII.  then  classified  by the order  of atoms o r m o l e c u l e s  processes  o f r e a c t i o n , t h a t i s , t h e number  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 the r a t e (or k i n e t i c s ) of the r e a c t i o n (Glasstone,  1948). Suppose before  a  irradiation,  i s the i n i t i a l and l e t x  a f t e r the l a p s e o f time maining t  be t h e d e c r e a s e o r c h a n g e  irradiation,  i n t h e g i v e n v o l u m e w i l l be  order.  The v a l u e s  of t  f r o m F i g u r e 17,  the r e a c t i o n i s o f  a n d a-x were c a l c u l a t e d  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  can be o b s e r v e d  t h e amount r e -  a-x. I f the p l o t o f  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 f i r s t and  t  c o n c e n t r a t i o n o f PABA  as F i g u r e 17.  the points f a l l  As  close t o a  - 75 -  TABLE X X I I .  Time o f 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 of I n c u No bation PABA  NonIrradiated PABA  Cone, of PABA micrograms p e r 10 ml. distilled wat e r  Time  72  0.018 0.854 0.015 0.824  0.056 0.046  0.854 0.921  Irradiation  Hours  1 48  of  2  3  4  100 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  100 50 10 2  0.770 0.638 0.569 0.509  0.367 0.092 0.081 0.076  0.620 0.215 0.131 0.125  0.201 0.076 0.066 0.061  F i g u r e s r e p r e s e n t g r o w t h o f A. s u b o x y d a n s . measured as O p t i c a l D e n s i t y .  -  straight line.  Since  PABA by u l t r a v i o l e t reaction, It  this  76  -  i s the case the a l t e r a t i o n of  irradiation  a p p e a r s t o be a f i r s t  order  i e . o n l y one m o l e c u l e 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 s o b v i o u s t h a t a d d i t i o n a l w o r k on t h i s  problem i s necessary. r e a c t i o n takes  phase o f t h e  I t m i g h t be p r o p o s e d t h a t t h e f o l l o w i n g  p l a c e by t h e a c t i o n of u l t r a v i o l e t  light  on  H  PABA:  fa  0 N  i  k  1st Reaction  Order.  Ultraviolet  Light *  I JJ u  cooH  C  N o r m a l PABA  o  o  H  A c t i v a t e d PABA  From t h e above two e x p e r i m e n t s i t seems s a f e t o c o n clude that the hypothesis  upon w h i c h t h e p r e s e n t  based i s sound i n p a r t a t l e a s t . that i r r a d i a t i o n no  longer  I t has been  demonstrated  o f PABA so a l t e r s t h e compound t h a t i t c a n  a c t as an " e s s e n t i a l m e t a b o l i t e "  under these  work i s  :  f o r A. s u b o x y d a n s  conditions.  Experiment 10:  To T e s t  the Influence  of U l t r a v i o l e t  Light  on t h e D i l u e n t o f t h e PABA S o l u t i o n . This violet ution.  light  e x p e r i m e n t was d e s i g n e d has an e f f e c t  t o determine i f u l t r a -  on t h e d i l u e n t o f t h e PABA  sol-  A washed m o t h e r c u l t u r e o f A. s u b o x y d a n s was  inoculated into  ( l ) 5 c c . b a s a l medium p l u s  s o l u t i o n t o make t h e f i n a l  concentration  5 c c . o f PABA  o f PABA 0.05  micro-  grams p e r 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  concentration  -  77 -  0.05 m i c r o g r a m s p e r 10 c c . medium; ( 3 ) 5 plus 5 c c . ofi r r a d i a t e d  distilled  c c . o f b a s a l medium  water, having  PABA t o t h e i r r a d i a t e d w a t e r a f t e r i r r a d i a t i o n final and  concentration  water.  t o make t h e  o f PABA 0.05 m i c r o g r a m s p e r 10 c c . medium;  5 c c . b a s a l medium p l u s  lastly  added n o r m a l  The m e d i a u s e d i n t h i s  5 cc. irradiated  distilled  e x p e r i m e n t were t h o s e o f  L a n d y and D i c k e n (1942) and L a n d y a n d S t r e i g h t o f f (1943), the  latter  having  (Appendix I V ) .  the a d d i t i o n o f purines  The r e s u l t s  obtained  i n t h e b a s a l medium  are presented  i n Table  XXIII. From t h e s e alters  r e s u l t s i t i s noted that u l t r a v i o l e t  PABA t o t h e e x t e n t  "essential metabolite"  t h a t i t c a n n o t be u s e d a s a n  f o r t h e g r o w t h o f A. s u b o x y d a n s a n d  does n o t a p p e a r t o a f f e c t t h e d i s t i l l e d that takes occurs  water.  Any r e a c t i o n  p l a c e when a s o l u t i o n o f PABA i s i r r a d i a t e d  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  molecule o fthe d i l u e n t .  not In the  I t i s worthy o f note that the  b a s a l medium o f L a n d y and S t r e i g h t o f f (1943)  fortified supports  light  l e s s g r o w t h o f A. s u b o x y d a n s when i t i s e n r i c h e d  w i t h n o r m a l PABA a s c o m p a r e d t o L a n d y and D i c k e n ' s medium. However w i t h t h e a d d i t i o n o f t h e p u r i n e s the  latter  irradiated  supports  t o t h e b a s a l medium,  greater growth of t h e t e s t  PABA o r i r r a d i a t e d d i s t i l l e d  o r g a n i s m when  w a t e r i s added t o  t h e medium. P r e v i o u s l y i t h a s b e e n s t a t e d ( B a u m g a r t n e r , 1936; Pratt,  1936) t h a t t h e a c t i o n o f u l t r a v i o l e t  light  on a s o l -  u t i o n a l t e r a its h y d r o g e n i o n c o n c e n t r a t i o n t o s u c h a d e g r e e  - 78 -  TABLE X X I I I .  The E f f e c t o f 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 o f A. s u b o x y d a n s .  Medium R e p l i - N o r m a l 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 cate PABA PABA Distilled Distilled" adiated No. Water p l u s Water Distilled Non-IrradWater i a t e d PABA Landy and Dicken  (1942)  Basal Medium w i t h no purines  1 2 3 4. 5  0.745 0.762 0.721 0.770 0.745  0.018  0.011 0.013 0.004 0.009  0.770 0.762 0.721 0.770 0.762  0.018  Av.  0.742  0.011  0.755  0.016  0.016  0.699 0.658 0.569 0.699 0.569  0.149 0.161 0.155 0.168  0.699 0.638 0.638 0.569 0.699  0.066 0.063 0.046 0.056 0.036  0.046 0.061 0.036 0.046 0.056  0.646  0.053  0.049  Landy and Streigh- ~ toff J  (1943)  Basal Medium plus purines  Av.  0.634  0.125 0.151  0.015 0.013 0.015 0.018  0.018  0.013 0.018 0.018  0.015  - 79 t h a t the  resulting  f o r most b a c t e r i a l resulting t h e n the  solution  has  growth.  However t h e y s t a t e  pH i s r e a d j u s t e d t o medium w i l l  a pH l e v e l w h i c h i s  s u i t the  observations a series  of  solutions,,  the  i n above e x p e r i m e n t , were i r r a d i a t e d  that i f  organism i n  s u p p o r t optimum g r o w t h .  the  question,  From t h e s e  same t h a t w e r e u s e d  i n order to  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 ion  unsuitable  hydrogen  concentration.  E x p e r i m e n t 10a  A series irradiation for  To D e t e r m i n e 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 H y d r o g e n Ion C o n c e n t r a t i o n o f a Solution. of  solutions  two  hours.  are The  subjected to  results  are  ultraviolet  presented  below.  TABLE X X I V .  on  The 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 t h e H y d r o g e n Ion C o n c e n t r a t i o n of a S o l u t i o n .  Distilled water PABA S o l u t i o n (,05 micrograms per 1 cc . )  From t h e s e r e s u l t s ion  c o n c e n t r a t i o n of  5.95  6.4  5.95  6.35  i t is  a solution  i r r a d i a t i o n , which c o n f i r m s the  gartner  (1936) and  pH A f t e r 2hour I r r a d i a t i o n  pH B e f o r e Irradiation  Solut ion  Pratt  (1936).  noted t h a t the is altered results  by  the  hydrogen ultraviolet  o b t a i n e d by  A f t e r the  Baum-  prescribed  - 80  -  amounts o f t h e s e s o l u t i o n s were added d e s c r i b e d i n E x p e r i m e n t 10,  t o t h e b a s a l medium as  the r e s u l t i n g  pH o f t h e medium  was c h e c k e d and r e a d j u s t e d , i f n e c e s s a r y , t o t h e l e v e l q u i r e d f o r optimum g r o w t h E x p e r i m e n t 10  o f A. s u b o x y d a n s .  reac-  The r e s u l t s o f  i n d i c a t e t h a t t h e change o f pH o f t h e 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  light  i s not the f a c t o r that  the growth response of t h e t e s t  organism, but t h e r e  reduces  exists  an a l t e r a t i o n o f t h e PABA m o l e c u l e t o s u c h a d e g r e e t h a t i t c a n n o t a c t as an " e s s e n t i a l m e t a b o l i t e " A.  suboxydans.  E x p e r i m e n t 11:  In t h i s  The A b i l i t y o f N o r m a l PABA t o O v e r - r i d e t h e I r r a d i a t e d PABA D u r i n g t h e G r o w t h o f A. s u b o x y d a n s . e x p e r i m e n t A. s u b o x y d a n s  ( l ) c o n t a i n i n g n o r m a l PABA, (2) and  (3)  containing irradiated  PABA a t a l e v e l 48  i n the n u t r i t i o n of  o f 0.05  hours of i n c u b a t i o n . The  containing  micrograms  p e r 10  After  60  p a r t o f t h e PABA had been a l t e r e d  after  normal  minutes of i r and l e s s  growth  Prolonged incubation  hours p e r m i t t e d n e a r l y normal growth.  by o n l y s l i g h t  normal  m l . o f medium  g r o w t h o b t a i n e d i n t h e b a s a l medium p l u s  of i r r a d i a t i o n  PABA,  The r e s u l t s a r e g i v e n i n T a b l e XXV.  was o b t a i n e d t h a n i n t h e n o r m a l c u l t u r e . f o r 96  irradiated  PABA t o w h i c h was added  PABA was e s s e n t i a l l y a s e x p e c t e d . radiation,  was grown i n m e d i a  A f t e r 90  minutes  most o f t h e PABA had b e e n a l t e r e d as e v i d e n c e d growth.  Even a f t e r 96  hours of i n c u b a t i o n ,  g r o w t h was. n o t c o m p l e t e l y n o r m a l 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. s u b o x y d a n s t o I r r a d i a t e d a n d N o n - I r r a d i a t e d PABA u n d e r V a r i o u s C o n d i t i o n s .  Growth Replicate No.  Growth Measured at H r s .  Normal PABA  i n  60 M i n u t e Irradiated PABA  0.770 0.783  1 2 3 4 5 6 7  48 48 48 48 48 96 96  0.955* 0.921 0.940 0.889 0.940 0.979 1.042  0.862  8 9 10  96 96 96  1.023 1.095 1.071  0.921 0.921 0.886  Added to. M e d i a a t 48 Hours  »  nil  Media  0.870  0.854 0.870 0.862  0.05 micrograms PABA  Containing  90 M i n u t e Irradiated PABA  No PABA  0.131 0.155 0.155 0.149 0.155 0.602 0.608  0.018 0.018 0.018  0.921 0.886 0.906  0..009  0.05 micrograms PABA  0.009 0.009  0.018  0..009 nil  0.018 0.018  F i g u r e s r e p r e s e n t g r o w t h o f A. s u b o x y d a n s measured as O p t i c a l D e n s i t y .  - 82 -  t h a n a t t h e end o f 48 h o u r s normal  of i n c u b a t i o n .  PABA a t 48 h o u r s t o r e p l i c a t e c u l t u r e s  t o grow n o r m a l l y i n t h e b a s a l permitted  the i n i t i a t i o n  of c e l l s  i n t h e now c o m p l e t e medium.  w h i c h has f a i l e d permits normal longer  medium p l u s  and d e v e l o p m e n t  e x p e r i m e n t has shown t h a t  the  The a d d i t i o n o f which  i r r a d i a t e d PABA, of the usual  growth.  irradiation  I n o t h e r words' t h i s  the a d d i t i o n  o f PABA t o a c u l t u r e  A subsequent  period,  experiment  i n which a  100 m i n u t e s , was u s e d , c o n f i r m e d  above r e s u l t s i n e n t i r e t y .  evident  that  o f .the p r e v i o u s e x p e r i m e n t s i t i s  PABA i s a l t e r e d by t h e a c t i o n  irradiation.  Since t h i s  of u l t r a v i o l e t  i s t h e case a s e r i e s of experiments  were c o n d u c t e d t o n o t e t h e e f f e c t o f u l t r a v i o l e t cells  violet  o f A_. s u b o x y d a n s . '  PABA, o r i t s enzyme s y s t e m s  microbial c e l l , absorption cidal  The g e r m i c i d a l  e n e r g y may be due t o t h e d e s t r u c t i o n  metabolite"  because  activity.  This  on t r y p t o p h a n e and t y r o s i n e  aminobenzoic  shown t h a t t h e of germi-  They s t u d i e d  acid restored cells.  t o be v e r y i n s i g n i f i c a n t ,  t h e growth This  that  of u l t r a v i o l e t i r -  statement appeared  however on t h e c o n t r a r y  very s i g n i f i c a n t observation.  the u l t r a -  i n r e l a t i o n to  o f b a c t e r i a , and s u m m a r i l y m e n t i o n e d  radiated microbial  of u l t r a -  f a c t seems t o have been o v e r l o o k e d by  violet  destruction  on  of the " e s s e n t i a l  w e l l i n the region  ( H a r r i s and H o y t , 1 9 1 9 ) .  action  activity  i t has b e e n p r e v i o u s l y  maximum o f PABA l i e s  light  on a n d w i t h i n t h e  e a r l y workers  the  number  t o grow i n t h e p r e s e n c e o f i r r a d i a t e d PABA  In t h e l i g h t  the  failed  i n print i t may be a  An e x p e r i m e n t was c o n d u c t e d .  -  83  t o n o t e i f PABA w o u l d r e s t o r e cells  -  the growth o f t h e i r r a d i a t e d  o f A_. s u b o x y d a n s .  Experiment 12:  The E f f e c t the  Washed c e l l s  of U l t r a v i o l e t  Growth  I r r a d i a t i o n on  o f A. s u b o x y d a n s .  o f A. s u b o x y d a n s were i r r a d i a t e d f o r  100 m i n u t e s u n d e r t h e 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 (Appendix V I ) .  These c e l l s w e r e t h e n i n o c u l a t e d  o f 0 i 0 5 m l . o f washed i r r a d i a t e d m o t h e r of media.  When t h e i r r a d i a t e d c e l l s  inoculated into was o b t a i n e d ,  medium e n r i c h e d  with  medium, no g r o w t h a f t e r Further  incubation  indicating  culture into  were  into the basal  o f PABA p e r 10 c c .  48 h o u r s o f i n c u b a t i o n  was a p p a r e n t .  t o 96 h o u r s r e s u l t e d i n n o r m a l  that the i r r a d i a t e d c e l l s  a series  o f PABA no g r o w t h  When i n o c u l a t e d  0.05 micrograms  at a l e v e l  o f A. s u b o x y d a n s  t h e b a s a l medium d e v o i d  as was e x p e c t e d .  Lamp  required  as  growth appreciable  t i m e t o r e t u r n t o t h e i r n o r m a l s t a t e i n t h e p r e s e n c e of. PABA. Irradiated  cells  micrograms  of i r r a d i a t e d  grow e v e n a f t e r  inoculated  into  t h e b a s a l medium p l u s 0 . 0 5  PABA p e r 10 c c . medium f a i l e d t o  96 h o u r s o f i n c u b a t i o n .  When n o r m a l PABA was  i  added a f t e r  96 h o u r s o f i n c u b a t i o n ,  t a i n e d a f t e r 48 h o u r s .  This  had t a k e n up t h e i r r a d i a t e d but, r e s t i n g period. sidered  n o r m a l g r o w t h was o b -  would suggest t h a t those  p r o d u c t and remained i n a v i a b l e  s t a g e f o r t h e p r e l i m i n a r y 96 h o u r  This  result  i n the light  cells  incubation  a p p e a r s s i g n i f i c a n t when i t i s c o n of i r r a d i a t e d c e l l s  inoculated  into a  - 84 -  PABA n e g a t i v e medium. grow d u r i n g t h e f i r s t  These l a t t e r  c e l l s not only f a i l e d t o  96 h o u r s o f i n c u b a t i o n b u t l i k e w i s e  d u r i n g a s u b s e q u e n t 48 h o u r s when 0.05 m i c r o g r a m s o f n o r m a l PABA p e r 10 c c . medium was added t o t h e medium. A 24 h o u r o l d w a s h e d m o t h e r for  a series  of time i n t e r v a l s .  of i r r a d i a t i o n ,  c u l t u r e was  irradiated  A t t h e end o f a t i m e l i m i t  0.05 m l . o f t h e s u s p e n s i o n o f 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, r a d i a t e d PABA and n o r m a l PABA.  The r e s u l t s  i r -  are presented  below. TABLE X X V I . The E f f e c t  B a s a l Medium plus  o f U l t r a v i o l e t L i g h t on t h e G r o w t h of k. s u b o x y d a n s .  Time of- I r r a d i a t i o n 0  No PABA 0.027* I r r a d i a t e d PABA 0.05 m i c r o g r a m s per 10 c c . medium 0.086 N o r m a l PABA; 0.05 m i c r o g r a m s per 10 cc medium 0.770  i n Minutes  20  40  60  80  0  0  0  0  0  0  0  100  120  0  0  0  0  0  0  0  0  0  0  0  x F i g u r e s r e p r e s e n t g r o w t h o f A. s u b o x y d a n s , m e a s u r e d 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 b y t h e d a t a t a b u l a t e d i n T a b l e X X V I , irradiation results of  o f a s u s p e n s i o n o f A. s u b o x y d a n s f o r 20 m i n u t e s  i n c o m p l e t e d e s t r u c t i o n and a l s o t h a t t h e a d d i t i o n  PABA t o i t s g r o w t h medium t o a l e v e l  o f 0.05  micrograms  - 85  -  per  10  the  i n o c u l a t e d media  were h e l d o v e r i n t h e i n c u b a t o r f o r  144  h o u r s , and s t i l l  no g r o w t h o f t h e t e s t  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.  organism  An e x p e r i m e n t s i m i l a r t o t h e above was i n w h i c h a 24  h o u r o l d washed c u l t u r e  exposed t o u l t r a v i o l e t minutes.  f o r 15,  s u s p e n s i o n was  number o f t h e v i a b l e c e l l s  interval. exhibited  A f t e r 15  out  o f A. s u b o x y d a n s  was  30,  45,  60  a p o r t i o n of the  r e m a i n i n g a f t e r each no g r o w t h  interest  A. s u b o x y d a n s . damaged beyond  destroyed.  agar (see Appendix I V ) .  This r e s u l t  gly-  a m o u n t s * o f PABA f o r t h e g r o w t h o f have been  t h e p o i n t where t h e a d d i t i o n o f PABA t o permit normal  o b t a i n e d i n E x p e r i m e n t 12.  their  growth.  The a b o v e o b s e r v a t i o n s do n o t c o n f i r m t h e  d a t a o f E x p e r i m e n t 12  i s of  T h i s medium i s  Apparently the irradiated c e l l s  g r o w t h menstruum w i l l  It i s felt,  however,  results that the  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 w o r k w h i c h i s p l a n n e d may The  ultra-  s i n c e t h e p l a t i n g medium u s e d was  known t o c o n t a i n a d e q u a t e  K  was  minutes of exposure t o  A. s u b o x y d a n s was  cerol yeast-extract  results.  time  on t h e a g a r 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  light,  particular  90  and  plated i n order to determine  minutes of i r r a d i a t i o n  e x p e r i m e n t i n t h a t a f t e r 15-20 violet  occurred.  carried  At t h e end o f e a c h t i m e i n t e r v a l ,  irradiated cell the  light  All  fact that the i r r a d i a t e d  confirm  such  organism i s i n o c u l a t e d  PABA c o n t e n t o f y e a s t - e x t r a c t ( B a c t o ) = 40 m i c r o g r a m s p e r gram ( L a n d y and D i c k e n , 1942)  -  into  -  l i q u i d m e d i a may be t h e f a c t o r  to remain i n a r e s t i n g available  stage u n t i l  to counteract  irradiation. are  86  that permits t h e organism a d e q u a t e n o r m a l PABA i s  the b i o l o g i c a l  action  of u l t r a v i o l e t  On t h e o t h e r h a n d , when t h e i r r a d i a t e d  seeded onto g l y c e r o l  great d i f f i c u l t y  yeast-extract  cells  a g a r , t h e y may have  t o o b t a i n t h e n o r m a l PABA a n d t h u s c a n n o t  overcome t h e a c t i o n o f t h e u l t r a v i o l e t  irradiation  previously  i m p o s e d on t h e m . R o b e r t s and A l d o u s (1949) s t a t e t h a t t h e a c t i o n o f ultraviolet  irradiation  on m i c r o b i a l  cells results  production o f a poison w i t h i n the c e l l conditions this  and t h a t u n d e r  p o i s o n c a n be i n a c t i v a t e d  or removed  allowing the c e l l  t o remain v i a b l e .  be  the irradiated  p r o d u c t o f PABA a n d t h a t  is  n o t removed, an i n h i b i t i o n o f c e l l  and  the cell  f i n a l l y dies.  i n the certain thus  This poison could i fthis  division  However i f t h i s  readily  product  i s created  product i s r e -  p l a c e d b y n o r m a l PABA, t h e n t h e o r g a n i s m c a n r e s u m e n o r m a l division  and g r o w t h . These w o r k e r s f o u n d a l s o t h a t r e c o v e r y o f b a c t e r i a  after  ultraviolet  irradiation  proceeds i n l i q u i d s  s t o p p e d o r a t l e a s t r e t a r d e d on s o l i d m e d i a . explanation f o r the c o n f l i c t i n g  VII.  results  The 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 In t h e p r e v i o u s  but i s  T h i s may be t h e  recorded  above.  L i g h t on PABA  section dealing with the 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 determinable  b y b i o l o g i c a l a s s a y u s i n g A. s u b o x y d a n s a s t h e  t e s t organism. logical  irradiated  PABA.  I t i s o f i n t e r e s t now t o c o n f i r m t h i s  f i n d i n g b y 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 .  achieve t h i s  curve  i r r a d i a t i o n as  bioTo  purpose t h e a b s o r p t i o n c u r v e s o f normal and  PABA w e r e d e t e r m i n e d  t o note  o f t h e i r r a d i a t e d compound d i f f e r s (  A l l spectrophotometric  i f the absorption from  data recorded  that of normal  b e l o w were o b -  t a i n e d 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 .  Experiment  13:  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 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 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 o f N o r m a l PABA  Curve  An a b s o r p t i o n c u r v e o f n o r m a l PABA (0.000036 M b u f f e r e d w i t h KHgPO^ a t pH 4.3) was d e t e r m i n e d , as F i g u r e 1 8 ( b ) f r o m t h e d a t a p r e s e n t e d Comparing t h i s  i n Table  and i s g i v e n XXVII.  absorption curve w i t h t h a t determined by  Rothman and H e n n i n g s e n (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 o f t h e t w o c u r v e s The  molecular  extinction coefficient  l i e a t 2785 A.  ( S ) o f t h e 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 c u r v e s h o w i n g an £ v a l u e o f 15,300, while the experimental curve The  shows an J v a l u e o f 13,900.  d i s c r e p a n c y between t h e p r e s e n t  figure  o f 13,900 and t h a t  o f 15,300 r e p o r t e d b y Rothman and H e n n i n g s e n c a n n o t b e accounted  f o r s i n c e these workers  do n o t 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 o f a 0.000036 M S o l u t i o n o f Normal. PABA when S u b j e c t e d t o V a r i o u s Wave L e n g t h s o f L i g h t .  Wave L e n g t h mp.  210 215 220 225 230 23 5 . 240 245 250 255 260 265 270 275 276  Optical  Density  0.322 0.331 0.321 0.255 0.165 0.105 0.094 0.131  0.188  0.265 0.345 0.416 0.465 0.490 0.495  Wave L e n g t h mu  277 278 278.5 279 280 285  290 295 300 305 310 315 320 325 330  Optical  Density  0.499 0.499 0.500 0.500 0.500 0.490 0.458 0.393 0.315 0.232 0.147 0.081  0.035 0.009 0.000  - 8.9  tinent  -  i n f o r m a t i o n s u c h a s s o l v e n t u s e d a n d t h e pH o f t h e i r  solutions.. i t i s known ( K u m l e r a n d S t r a i g h t , 1943) t h a t t h e  Since  pH a t w h i c h t h e a b s o r p t i o n the  p o s i t i o n of the curve  as t h e m o l e c u l a r the as ous  discrepancy  i s determined i n f l u e n c e s  with respect  t o w a v e l e n g t h as w e l l  extinction coefficient,  i ti s felt  noted i s not too s e r i o u s .  c a n be seen f r o m F i g u r e  that  I t i s also possible  19 t h a t PABA a t pH 4.3 i n  s o l u t i o n does n o t obey t h e L a m b e r t - B e e r Law.  true then t h e molecular be  curve  aque-  I f this i s  extinction coefficient w i l l  tend t o  a f u n c t i o n , o f c o n c e n t r a t i o n o f t h e s o l u t e a n d h e n c e 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 (b)  values.  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 I r r a d i a t e d PABA.  Curve o f  Two s o l u t i o n s o f PABA, 0.0036 and 0.000036 M, buffered with  KH^PO^ a t pH 4.3, w e r e i r r a d i a t e d  After t h i s period of i r r a d i a t i o n , the former d i l u t e d t o make t h e c o n c e n t r a t i o n absorption  curves  of both  s o l u t i o n was  o f PABA 0.000036 M. The  s o l u t i o n s were d e t e r m i n e d and a r e  g i v e n a s F i g u r e 18 ( c ) a n d 18 ( d ) f r o m t h e d a t a in  Tables  XXVIII  The concentrated  f o r 4 hours.  tabulated  and X X I X .  molecular  e x t i n c t i o n c o e f f i c i e n t o f t h e more  irradiated  11,000 ( F i g u r e 18 ( c ) ) . that approximately  PABA s o l u t i o n was c a l c u l a t e d t o be From t h i s i L v a l u e  21 per c e n t  was a l t e r e d b y t h e u l t r a v i o l e t  i t was c a l c u l a t e d  o f t h e 0.0036 M PABA s o l u t i o n light.  - 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 L e n g t h s o f L i g h t a t _a Concent r a t i o n o f  0.000036 M.  Wave L e n g t h mu  210 215 220 225 230 235 240 245 250 255 260  Optical'Density  0.277  0.288  0.268 0.214 0.139 0.094 0.086  0.112 0.157 0.215 0.278  Wave L e n g t h mu  265 270 275  278 280 285  290 300 310 320 330  Optical  Density  0.335 0.371 0.395 0.396 0.396 0.385 0.360 0.254 0.120 0.033 0.004  - 91 -  TABLE X X I X .  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 L e n g t h s o f L i g h t .  Wave L e n g t h mu 210 220  225  230  235  240  245 253 260  270 275 278.5  Optical  Density  0.415 0.348 0.304 0.260 0.222 0.200 0.192 0.197  0.204 0.196 0.181 0.170  .  Wave L e n g t h nyi  Optical  Density  279 280 285 290 295  0.167 0.166 0.149 0.142 0.136  305 310 315 320  0.120 0.110 0.102 0.092 0.086 0.080  300  325  330  0.130  - 92 -  The  absorption  curve  PABA a p p e a r s t o be v e r y previous  curves.  o f t h e more d i l u t e d  different  ( F i g u r e 18 ( d ) ) t o t h e  From t h e work o f Doub and V a n d e n b e l t  the a b s o r p t i o n  curve  18 ( d ) i s v e r y  s i m i l a r t o the absorption  Since  s o l u t i o n of  t h i s appears  of i r r a d i a t e d  PABA p r e s e n t e d curve  t o be t h e c a s e i t i s l i k e l y  (1947)  as F i g u r e  of  phenol.  that the  amino g r o u p o f the PABA m o l e c u l e  has been  action  or r a t h e r a l l o f the normal  of the u l t r a v i o l e t  light,  removed by t h e  PABA i n t h e 0.000036 M s o l u t i o n has been a l t e r e d o r c h a n g e d to another It Electric cent  compound b y t h e u l t r a v i o l e t has been shown e a r l i e r  Germicidal  absorption  compound w i l l  Lamp u s e d t o i r r a d i a t e  curves  absorb  i s suggested that  t o use monochromatic maximum of' PABA. that  PABA e m i t s  o f 2537 X.  at t h i s  wave l e n g t h ,  light  If this  o f 2785 X w h i c h i s carried  Electric  of u l t r a v i o l e t  Figure  i s the  densities  therefore  be e x p e c t e d  specifically  Lamp i s u s e d as  light.  19 i n d i c a t e s t h a t t h e r e  o f 2790 8 t h e r e  creasing  this  absorption  o u t , i t would  Germicidal  PABA obeys the L a m b e r t - B e e r Law. length  90 p e r  i n f u t u r e w o r k i t w o u l d be a d v i s a b l e  PABA would be a l t e r e d more r a p i d l y and more  the source  General  From t h e  o f PABA i t i s i n d i c a t e d t h a t  some l i g h t  t h a n when t h e G e n e r a l  then  ( F i g u r e 2) t h a t t h e  o f i t s r a d i a t i o n a t a wave l e n g t h  given  it  irradiation.  This  i s a possibility shows t h a t  that  a t a wave  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 -  solution concentration  o f PABA and t h e o p t i c a l  of t h e s o l u t i o n s ( s e e T a b l e  XXX).  If this  i s true  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 t h e  - 93 -  TABLE XXX.  The P r o p o r t i o n a l i t y B e t w e e n I n c r e a s i n g ; C o n c e n t r a t i o n o f 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 o f PABA i n micrograms per c c . d i s t i l l e d water  0 0.5 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0  Optical  Density  0.000 0.041 0.075 0.166 0.252 0.366 0.458 0.515 0.662 0.700 0.781 0.875  - 94  -  d e t e r m i n a t i o n o f PABA.  VIII.  P r e l i m i n a r y Tests t o Isolate the I r r a d i a t e d of PABA. P r e l i m i n a r y attempts  r a d i a t e d product  or p r o d u c t s  Products  were made t o i d e n t i f y t h e i r o f PABA.  A s o l u t i o n of normal  PABA a t a c o n c e n t r a t i o n o f 500 m i c r o g r a m s o f PABA p e r c c . d i s t i l l e d w a t e r was e x p o s e d 8 hours.  After this  to ultraviolet  irradiation for  p e r i o d i t was n o t e d t h a t t h e s o l u t i o n  had t u r n e d a d a r k brown a n d showed e v i d e n c e o f a b r o w n p r e cipitate. resulting  The d i l u e n t was d i s t i l l e d  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  I t was f o u n d  that this  r e s i d u e was s l i g h t l y  per c e n t a l c o h o l , a c e t o n e in  o f f i n vacuum and t h e  e t h e r and c a r b o n  and d i s t i l l e d  tests.  s o l u b l e i n 95  water;  and i n s o l u b l e  tetrachloride.  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 t h e i d e n t i t y o f the i r r a d i a t e d product  o f PABA, h o w e v e r 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 pounds i n a p u r e  in isolating  t h e compound o r com-  s t a t e a n d t h e n , w i t h t h e a i d of c h e m i c a l  tests, the identity  o f t h e s e compounds may be e s t a b l i s h e d .  - 95 -  SUMMARY (1)  T h i s work  has been b a s e d on a w o r k i n g  hypothesis  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 violet  light  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  e n e r g y i n t h e u l t r a v i o l e t r e g i o n by one o r more e s sential  cellular  metabolites.  I t i s further  t h a t the a b s o r p t i o n produces a molecular absorbing  metabolite  of such a n a t u r e  then (2)  quite similar different  Bacteriostasis  ensues.  associated with the b i o l o g i c a l and t h e s t u d y  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  in this (3)  has been r e v i e w e d  to support  and i s p r e s e n t e d  subjects t h e above  by s e c t i o n s  thesis.  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  cedure f o r t h e d e t e r m i n a t i o n was done  action  of p-aminobenzoic  a c i d and s u l p h o n a m i d e s as w e l l as o t h e r r e l a t e d  hypothesis  pre-  to i n t e r f e r e w i t h t h e  f o l l o w s and u l t i m a t e l y d e a t h  of u l t r a v i o l e t l i g h t  acid)  while  to i t s metabolic  f u n c t i o n s of the c e l l .  The l i t e r a t u r e  i n the  ( i n t h i s case p-aminobenzoic  cursor i s s u f f i c i e n t l y normal m e t a b o l i c  shift  t h a t t h e a l t e r e d compound  structurally s t i l l  suggested  of p-aminobenzoic  pro-  acid  i n order to detect 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 r o d u c e an a l t e r a t i o n  i n the p-aminobenzoic  acid  molecule. (4)  A d i s c u s s i o n o f the growth requirements  of the t e s t  -  organism, is  96  A c e t o b a c t e r s u b o x y d a n s , has  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 procedure of p-aminobenzoic found  a c i d has  necessary t o modify  I t was  suggested  b e e n i n c l u d e d and  f o r the determination  been recommended, as i t was the p u b l i s h e d assay  t h a t t h i s recommendation s h o u l d  f o l l o w e d i n f u t u r e work a l o n g t h e l i n e s mentations (6)  procedures,,  of t h e  i r r a d i a t i o n i t seems s a f e t o c o n c l u d e t h e s i s upon w h i c h i n part,  irradiation that  experi-  reported herein.  I n t h e s t u d y o f t h e b i o l o g i c a l a c t i o n of  least  be  that the  the p r e s e n t work i s based  i n t h a t i t has  of p-aminobenzoic  ultraviolet hypo-  i s sound, a t  been demonstrated a c i d so a l t e r s  i t c a n no l o n g e r a c t as an  that  the  compound  "essential metabolite"  f o r A c e t o b a c t e r suboxydans, jinder  the p r e v a i l i n g  con-  ditions. E x p e r i m e n t a t i o n s were c a r r i e d of u l t r a v i o l e t s u b o x y d a n s , and  irradiation  on t h e  the subsequent  out t o show t h e  c e l l s of Acetobacter  study  to observe  " e s s e n t i a l m e t a b o l i t e " , p-aminobenzoic t o c o u n t e r a c t t h e a c t i o n s of t h e s e (7)  effect  i f the  a c i d , was  able  irradiations.  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 on p - a m i n o b e n z o i c  a c i d was  included.  the a b s o r p t i o n curves of normal aminobenzoic  a c i d were c a r r i e d  and  D e t e r m i n a t i o n s of irradiated  out w i t h the  Beckman M o d e l DU S p e c t r o p h o t o m e t e r . the a b s o r p t i o n curve of i r r a d i a t e d  light  I t was  p-  aid of  the  shown t h a t  p-aminobenzoic  acid.  *  -  (7)  (continued):  -  d i f f e r e d markedly from that o f normal  p-aminobenzoic (8)  97  acid.  P r e l i m i n a r y attempts to i s o l a t e d u c t or p r o d u c t s o f p - a m i n o b e n z o i c  the i r r a d i a t i o n  pro-  a c i d were made.  Various s o l u b i l i t y  tests  o f t h e new  compound were  carried  out and r e c o r d e d .  I t i s impossible at present t o state  t h e i d e n t i t y of t h e i r r a d i a t i o n p r o d u c t o f p-aminobenzoic acid, ful  however f u t u r e work w h i c h i s p l a n n e d may  i n i s o l a t i n g t h e compound  s t a t e and w i t h t h i s may (9)  be  result  o r compounds  the i d e n t i t y  i n a pure  of these  compounds  established.  The o r i g i n a l  h y p o t h e s i s emphasized  that the i r r a d i a t i o n  the l i k e l i h o o d  product of p-aminobenzoic  w o u l d a c t as a n a n t i - m e t a b o l i t e . not  be s u c c e s s -  The w o r k r e p o r t e d  support such a c o n t e n t i o n i n e n t i r e t y .  suggest that  e i t h e r an a n t i - m e t a b o l i t e  does  I t does  i s formed or  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 " , benzoic a c i d ,  acid  p-amino-  i s d e s t r o y e d by i r r a d i a t i o n a s 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  assay.  - 98 -  APPENDIX I .  Review o f P h o t o c h e m i c a l  The occurs  during  ascribed of  loss  ( H e y r o t h , 1941j G l a s s t o n e , 1948)  o f r a d i a n t energy  i t s passage  o f a beam o f l i g h t  through  to three processes;  a system,  ticles  Reactions  (b) s c a t t e r i n g  a gas o r a l i q u i d i s  (a) r e f l e x i o n of t h e l i g h t  i n t h e gas o r b y c o l l o i d a l  ( c ) a b s o r p t i o n by t h e m o l e c u l e s  The  last  three  ph.otochemically •  Consequently  such  of the l i g h t  cally  absorbed  emission the  law.  i n producing  absorbed  as l i g h t  be some r e -  and t h e c h e m i c a l  reaction.  During the  T h i s law s t a t e s t h a t by t h e r e a c t i n g  chemical  the absorbed  change."  which occurs  Not chemi-  radiation  o f t h e same or a n o t h e r  of r a d i a n t energy  absorption.  importance  by a s y s t e m i s e f f e c t i v e  b e c a u s e i n some c a s e s  re-emitted  should  r a d i a t i o n s which are absorbed  system are e f f e c t i v e all  i n the l i q u i d ,  a c o n n e c t i o n was g e n e r a l i z e d i n t e r m s  known as t h e G r o t t h u s - D r a p e r "only those  par-  o f t h e gas or l i q u i d .  there  change o c c u r r i n g i n a p h o t o c h e m i c a l century  by a n y d u s t  i s t h e o n l y one o f  l a t i o n s h i p between t h e l i g h t  19th  at the surfaces  particles  and  of these  which  may be  frequency,  an  simultaneously  This re-emission of l i g h t  has b e e n  with  termed  fluorescence. Lambert  i n 1760  between t h e e x t e n t  showed t h a t a r e l a t i o n s h i p  of l i g h t  existed  a b s o r p t i o n and t h e d e p t h o r  _ 99 -  thickness put  of the absorbing system.  This  r e l a t i o n s h i p was  i n t h e f o l l o w i n g f o r m c a l l e d t h e L a m b e r t ' s Law:  fractions cessive  "equal  o f t h e i n c i d e n t r a d i a t i o n a r e a b s o r b e d by s u c -  layers  of equal t h i c k n e s s  of t h e 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 c a n be f o r m e d :  where I and I intensities,  denote t h e t r a n s m i t t e d  and i n c i d e n t  1 i n cm. i s t h e t h i c k n e s s  light  of the absorbing  l a y e r , a n d k, t h e c o n s t a n t w h i c h i s c h a r a c t e r i s t i c o f t h e latter.  This  coefficient light  c o n s t a n t i s r e f e r r e d t o as t h e  of the r e a c t i n g  o f a p a r t i c u l a r wave l e n g t h ,  varies with  t h e wave l e n g t h  From t h e e q u a t i o n g i v e n absorbed  I t has r e f e r e n c e  to  and t h e v a l u e o f "k"  of the absorbed r a d i a t i o n .  above t h e i n t e n s i t y o f t h e l i g h t  ( l a b s ) i s equal t o t h e d i f f e r e n c e between t h e  intensities light;  system.  absorption  of the incident  ( l ) and t h e t r a n s m i t t e d ( I ) Q  therefore: labs  = I  Q  =1 = I  - I - (I  - k l  )  (1 - e - k l )  0  Where t h e 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  c a s e o f PABA d i s s o l v e d  i n a suitable solvent,  the r e -  l a t i o n s h i p b e t w e e n t h e 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 given  i n t h e f o r m o f B e e r ' s Law.  mathematical representation  of t h i s  I * I-  0  i o -  The  l a w i s as f o l l o w s : e  c  l  - 100  -  where "e" i s t h e 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 " t h e c o n c e n t r a t i o n o f t h e a b s o r b i n g A c c o r d i n g t o t h e Quantum a n d . E i n s t e i n , a body c a n n o t  T h e o r y as g i v e n b y or absorb  a continuous  or  g i v e n o u t as i n t e g r a l m u l t i p l e s o f a d e f i n i t e  for  as a quantum.  The e n e r g y  radiant  in  known  manner.  emit  then  Planck energy  c a n o n l y be t a k e n  I f "E" i s t h e e n e r g y  a p a r t i c u l a r r a d i a t i o n frequency,  second,  component.  up  amount,  o f t h e quantum  Y i n v i b r a t i o n s per  t h e 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  P l a n k ' s c o n s t a n t , e q u a l t o 6.62 Einstein ization  (1912, 1913)  i n photochemistry  chemical Equivalent.  x 10"^  erg sec.  i n t r o d u c e d an i m p o r t a n t  i n t h e form  of t h e l a w of  This law states, t h a t "each  t a k i n g part i n a chemical r a c t i o n , which sult  of t h e a b s o r p t i o n of l i g h t ,  the r a d i a t i o n  vibrations  per s e c o n d ,  e q u a l t o hV.  x 10 3. 2  Photo-  molecule  i s a direct re-  This law i m p l i e s  of t h e absorbed  radiation i n  t h e n t h e c o r r e s p o n d i n g quantum i s  Therefore  the energy,  "E", a b s o r b e d  i s t h e n NhY, where "N" i s t h e A v o g r a d o number 6.06  general  t a k e s up one quantum o f  causing the r e a c t i o n " .  t h a t i f -V i s t h e f r e q u e n c y  as  per mole  equal t o  Hence we have t h e r e p r e s e n t a t i o n a s : E = NhV e r g s p e r m o l e .  The f r e q u e n c y per  second  light  o r 3.0  "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  i s e q u a l t o c/)^ , w h e r e " c " i s t h e v e l o c i t y o f x 10  10  cm. p e r s e c o n d  and \ i s t h e wave l e n g t h  - 101-  in  cm.  However t h e l a t t e r  ftngstom  i s more f r e q u e n t l y e x p r e s s e d i n  u n i t s , where 1A = 1 0 ~  c a n be w r i t t e n  cm. and t h u s an e x p r e s s i o n  8  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 c a n now be  written:  Q  E = Nhc x 10  X  The q u a n t i t y E w h i c h reacting  substance  of r a d i a t i o n  e r g s p e r mole  i s the energy  absorbed  p e r mole o f  i s u s u a l l y r e f e r r e d t o as one  einstein  o f 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 o r quantum y i e l d , d e f i n e d as t h e number stance that reacts  of moles of the l i g h t  f o r each  which i s  absorbing  e i n s t e i n of absorbed  sub-  radiation  i s v e r y i m p o r t a n t as f a r as t h e r e s u l t  of a photochemical  r e a c t i o n i s concerned.  t h e quantum  For convenience  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  efficiency  ( y i e l d ) = Number o f m o l e s r e a c t i n g Number o f e i n s t e i n s  absorbed  By t h e Law o f 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 viously, system  i t would  be e x p e c t e d  that  i f the reaction  i s one a s s o c i a t e d o n l y w i t h t h e a c t i o n  t h e n t h e quantum e f f i c i e n c y w o u l d many c a s e s t h i s t h a t the f i r s t  be u n i t y .  i s not the a c t u a l r e s u l t .  of l i g h t , However i n  I t was  suggested  step of the photochemical r e a c t i o n ,  a b s o r p t i o n of l i g h t ,  i e ,the  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 properly pertains.  of a  process i n which  the E i n s t e i n law  I n t h i s p r i m a r y p r o c e s s each  molecule  -  a b s o r b s one  102  quantum, h o w e v e r , a f t e r  such a b s o r p t i o n ,  a  secondary process occurs i n which the molecules of r e actant take part. be q u i t e c o m p l e x , yield  T h i s subsequent and w i l l ,  secondary process  in fact,  cause the  of the o v e r a l l r e a c t i o n t o v a r y from  may  quantum  unity.  - 103 -  II.  APPENDIX The Use o f M i c r o o r g a n i s m s t o As s a y  During  the past  few years  Vitamins  v a r i o u s methods h a v e b e e n  d e v e l o p e d and e m p l o y e d 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 content.  P h y s i c a l and c h e m i c a l  ever  are i n c o n t e s t a b l e only i f the r e s u l t s  they  from these cedures.  methods a r e u s e f u l , howobtained  methods c a n 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 - ' As a r e s u l t  b i o l o g i c a l methods have b e e n  u s e f u l i n the discovery criteria  of v i t a m i n s  for qualitative  as w e l l a s b e i n g standardized  and  extremely  and a l s o as t h e f i n a l  and q u a n t i t a t i v e v i t a m i n  assays  a means w h e r e b y o t h e r methods c a n be evaluated.  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 the  vitamin  s u c h as  r a t a r e o f t e n c o s t l y and t i m e c o n s u m i n g , and u s u a l l y  c a n n o t be e m p l o y e d when s m a l l amounts o f 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 . require c e r t a i n vitamins  The d i s c o v e r y t h a t  microorganisms  f o r g r o w t h has p r o v i d e d  a  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 .  new, For  example an o r g a n i s m w h i c h r e q u i r e s a g i v e n v i t a m i n i s u s e d , and  a l l other  i n t h e medium.  f a c t o r s necessary A standard  f o r growth are kept  curve  i s then obtained  constant  by  v a r y i n g the c o n c e n t r a t i o n of the v i t a m i n i n question plotting  t h e g r o w t h r e s p o n s e i n t h e c u l t u r e medium  the v a r i o u s  concentrations  of the v i t a m i n .  a g i v e n v i t a m i n i n an unknown  and  against  The amount o f  s u b s t a n c e c a n t h e n be  - 104 -  d e t 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 o f t h e known vitamin.  A comparison  of t h e r e s u l t s from t h i s  substance  a g a i n s t t h e 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 question w i l l  show t h e amount o f tha(6 f a c t o r i n t h e u n -  known. T h i s method o f 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 favor. to  gaining  A t t h e p r e s e n t t i m e i t s u s e a p p e a r s t o be r e s t r i c t e d  t h e B v i t a m i n s , , however as t i m e p r o g r e s s e s , s p e c i a l  tests w i l l the  u n d o u b t e l y be d e v e l o p e d f o r a l l t h e members o f  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  organisms  t h e 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 ,  micro-  were used t o d e t e r m i n e  o f PABA  i f any a l t e r a t i o n  r e s u l t s when t h a t s u b s t a n c e i s e x p o s e d  to ultraviolet  radiations. The exists  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 there  a s u i t a b l e m i c r o o r g a n i s m w h i c h r e q u i r e s PABA f o r i t s  normal growth.  Since t h i s  extremely u s e f u l ,  survey of the l i t e r a t u r e  i t i s wise at t h i s  proved  point to review i t  briefly. The  Growth Promoting A c t i v i t i e s  (i)  o f PABA f o r B a c t e r i a ;  M i c r o o r g a n i s m s t h a t do n o t r e q u i r e t h e a d d i t i o n o f PABA t o t h e i r g r o w t h medium. Woods ( 1 9 4 0 ) r e p o r t e d t h a t  effect  PABA h a d no  on t h e r a t e o r mass o f g r o w t h  haemolyticus or Bacterium c o l i conditions  significant  of Streptococcus  under t h e p r e v a i l i n g  test  s i n c e i t was n o t n e c e s s a r y t o a d d 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  investigators  have shown t h a t PABA does n o t s t i m u l a t e t h e g r o w t h o f  - 105 -  Streptococcus  1941) S t a p h y l o c o c c u s  viridans (Miller,  and J e r m a t a , 1941) o r Pneumococcus ( S t r a u s e ,  (Spink  aureus et a l .  1941) and a l s o t h a t i t h a s n o t b e e n f o u n d e s s e n t i a l f o r other  pathogenic  b a c t e r i a ( F o x , 1942).  organisms are able  Apparently  t o synthesize t h i s f a c t o r from  these other  precursors. 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 , that  the fact  i t i s a g r o w t h f a c t o r f o r a f e w o r g a n i s m s s h o u l d be  supplemented by e v 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 , that i s t o prove that i t i s synthesized  by o r g a n i s m s a b l e t o grow i n P A B A - f r e e c u l t u r e  media. L a n d y e t a l (1943), w h i l e  studying  bacterial  synthesis  o f PABA, c u l t u r e d a g r e a t v a r i e t y o f o r g a n i s m s i n a PABAf 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  synthesize  PABA i n r e a d i l y m e a s u r a b l e a m o u n t s , and a l s o t h a t i n most instances the bulk medium.  o f PABA p r o d u c e d i s f o u n d i n t h e c u l t u r e  Few o f t h e s p e c i e s r e t a i n  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  e l a b o r a t e PABA i s n o t i n t e n d e d comparison of various that  strains  any g r e a t  amount o f  of b a c t e r i a t o  t o convey any q u a n t i t a t i v e  o r g a n i s m s , b e c a u s e i t was  of a given  species  c u l t u r e d under  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 amount o f PABA.  observed apparently t h e same  T h i s p o i n t i s shown i n t h e c o n d e n s e d t a b l e  g i v e n b y L a n d y 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 o f P_«_ a m i n o b e n z o i c A c i d ( L a n d y e t a l . 1943)  Organism  p-Aminobenzoic A c i d .found ( m i c r o g r a m s p e r cc . c u l t u r e ) In C e l l s In Medium T o t a l  37 37 37 37 30 37 37 37 37 30  0 0.038 0 0 0 0.070 0.004 0.008  0.019 0.007 0.171 0.063 0.004 0.016 0  l e n d s s u p p o r t t o Wood's t h e s i s  that the  sulphonamide i n h i b i t i o n  24 24 72  24 24  24 24 24 72 72  -  0  It is  (1943) t h a t t h e " s e n s i t i -  o f 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 w h e t h e r  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 o r n o t .  An o r g a n i s m 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 w o u l d be more s e n -  s i t i v e t o s u l p h o n a m i d e s t h a n ones w i t h g r e a t e r powers.  The f i n d i n g  synthetic  that c e r t a i n bacteria synthesize  ( T a b l e A ) a p p e a r s t o make t h i s In  0.013  i s due t o PABA i n t e r f e r e n c e .  s u g g e s t e d by Landy and h i s c o l l e a g u e s vity"  0.018 0.007  0. 018 0.045 0.013 0.019 0.007 0.241 0.067 0.012 0.016 0  Shigella dysenteriae Escherichia c o l i Brucella abortus Bacillus subtilis B a c i l l u s megatherium C o r y n e b a c t e r i u m diphtheriae Staphylococcus aureus Streptococcus hemolyticus Lactobacillus casei L a c t o b a c i l l u s arabinosus This f i n d i n g  Incubation °C Hr.  suggestion a d e f i n i t e  a n y c a s e 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  PABA  possibility.  by a g i v e n  o r g a n i s m w o u l d p l a y some p a r t i n t h e d e g r e e o f i t s r e s i s t a n c e to  sulphonamide  action.  - 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 t h e a d d i t i o n o f PABA t o t h e i r g r o w t h medium It  has been p o i n t e d o u t ( a b o v e ) t h a t t h e m a j o r i t y o f  b a c t e r i a c a n s y n t h e s i z e PABA, w h i l e a f e w may and do r e q u i r e the  presence of t h i s  ment and d i v i s i o n .  a r o m a t i c amine f o r n o r m a l c e l l This simple organic  f o r growth by A c e t o b a c t e r suboxydans where 0 . 0 5 m i c r o g r a m s maximum g r o w t h . factor  (Lampen, e t a l . 1942)  o f PABA p e r t e n c c . medium s u p p o r t s growth  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 s u b (isbell,  1942} L e w i s , 1942j Shankman,  I s b e l l (1942) f o u n d 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 also that the  compound i s n e e d e d  T h a t t h i s a r o m a t i c amine i s a t r u e  sequent i n v e s t i g a t o r s 1943).  develop-  o f PABA p e r t e n c c . medium and  t h i s growth f a c t o r i s 10,000  n e x t most a c t i v e compound.  t h a t t h e r e a r e two s t r a i n s  times as a c t i v e as  Shankman (1943) o b s e r v e d  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 , w h i c h i s a m u t a n t ,  does  not. 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 . b y C l o s t r i d i u m b u t y l i c u m No. 2 8 , a n d b y C l o s t r i d i u m felsineum. In t h e presence of b i o t i n micrograms  (1.5  x 1 0 - 4 t o 1.5  x 10""°  p e r «c. o f medium) PABA f u n c t i o n s a s a g r o w t h  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 l o w as 1 x 10"^ m i c r o g r a m s  p e r 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 a n d M i t c h e l l , T  1944)  Coryne-  - 108 -  bacterium diphtheriae  (Chattaway,  et a l . ,  1942) and  N e u r o s p o r a m u t a n t (Tatum a n d B e a d l e , 1 9 4 2 ) w e r e 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 that t h e e f f e c t i v e n e s s  of  PABA a s a g r o w t h  of  Tatum a n d B e a d l e ( 1 9 4 2 ) d e c r e a s e s w i t h t h e i n c r e a s e i n pH  'of  thenutrient  f a c t o r f o r t h e Neurospora  s o l u t i o n , thus i n d i c a t i n g  m o l e c u l a r and n o t t h e i o n i c amine. (Wyss e t a l . , It  a c t i v i t y forthe  form of t h e simple aromatic  1944).  h a s been d e m o n s t r a t e d  by L e u c o n o s t o c  c r a s s a mutant  mesenteroides  that-PABA  i salso  required  Pd-60 ( 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 , 1 9 4 6 j 1 9 4 6 a ) a n d s t i m u l a t e s growth  of C I . b o t u l i n u m ( R o e s s l e r and Brewer,  l y Rainbow (1948) n o t i c e d t h a t two s i n g l e f e r m e n t a t i o n y e a s t s ('Yeast presence  sulphonamides  cell  Recent-  s t r a i n s of  and 'Yeast 4 7 ' )  o f PABA i n t h e medium f o r optimum Since t h e growth  of  45'  1946).  requires the  growth.  i n h i b i t o r y action of the majority  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 b y  PABA, i t i s l i k e l y t h a t t h e e l u c i d a t i o n o f t h e m e t a b o l i c function of t h i s action that  of these drugs.  sulphonamides  involved was is  factor w i l l  i l l u m i n a t e t h e b a s i c mode o f  I t has b e e n s u g g e s t e d  competitively i n h i b i t  i nthe u t i l i z a t i o n  o f PABA.  (Woods, 1 9 4 0 )  t h e enzyme  reaction  Since this suggestion  proposed i n f o r m a t i o n as t o t h e n a t u r e o f t h i s  utilization  a t hand (Lampen a n d J o n e s , 1 9 4 7 ) a n d t h e i n t e r r e l a t i o n -  s h i p s o f 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 a n d " f o l i c are  of p a r t i c u l a r  interest.  acid"  -  It  1 0 9  -  may a p p e a r 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 about a growth i n h i b i t o r y inhibitory  action that  a c t i o n of sulphonamides.  compound w i l l  bring  i s s i m i l a r t o that With t h i s  i n mind,  then  i t may p o s s i b l y be t h e f a c t t h a t t h e n e w l y f o r m e d compound will  competitively  volved  inhibit  an enzyme r e a c t i o n t h a t  i n the u t i l i z a t i o n  is i n -  o f PABA  M i c r o b i o l o g i c a l and C h e m i c a l Methods f o r t h e D e t e r m i n a t i o n o f PABA (i)  M i c r o b i o l o g i c a l Methods; A semi-quantitative  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  b y Rubbo e t a l . ( 1 9 4 1 )  butylicum.  O t h e r more p r e c i s e methods s i n c e have been  developed using  Dicken  (1942)  determination.  colorimeter  1944).  Landy and  They m e a s u r e i n a  t o PABA l e v e l i n t h e -  This t e s t i s h i g h l y s p e c i f i c ,  o f PABA and o t h e r  since the isomers  r e l a t e d s u b s t a n c e s were f o u n d t o have no  biological activity.  I n 1945,  a modification i n the  A c e t o b a c t e r s u b o x y d a n s a s s a y f o r PABA was ( C h e l d e l i n and Bennett, 1 9 4 5 ) . several respects added PABA*  determination.  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 medium.  aceto-  have employed A c e t o b a c t e r suboxydans f o r t h e  organism i n t h i s  photoelectric  Clostridium  t h e same m i c r o o r g a n i s m f o r t h i s  and Wood, 1 9 4 2 ; Lampen a n d P e t e r s o n ,  (Park  test  using  published  The medium was c h a n g e d i n  t o permit greater  growth response t o  - 110 -  Lewis  (1942) d e s c r i b e d a method w h e r e b y L a c t o -  b a c i l l u s a r a b i n o s u s was u s e d  i n the  M i t c h e l l and h i s a s s o c i a t e s (1943)  m i c r o b i o l o g i c a l assay.  made use o f t h e N e u r o s p o r a  (1942).  as t h e t e s t o r g a n i s m  crasrea m u t a n t o f Tatum and  The l a t t e r method was r e v i s e d b y t h e same  e t a l . (1943) f o u n d t h a t s t r o n g a c i d  or alkaline  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 t h e c o r r e c t of n a t u r a l s u b s t a n c e s  workers  (Thompson e t a l . 1943).  i n a more r e c e n t p u b l i c a t i o n Thompson  Beadle  PABA c o n t e n t  s i n c e i t o c c u r s i n f r e e a n d bound  f o r m s ( B l a n c h a r d , 1941).  However a s i t i s a p p a r e n t  that  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 a n e x t r e m e l y r e s i s t a n t combination, resistant  p a r t o f t h e PABA i s i n a compound  to acid hydrolysis.  R e c o m m e n d a t i o n s were made  t o f r e e PABA f r o m i t s bound f o r m b y u s i n g 5N NaOH a n d  sub-  j e c t i n g t h e 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 h o u r (Lampen and P e t e r s o n , 1944).  (ii)  Chemical  Methods r  Using thiamine method was d e v e l o p e d  (B-^) a s a r e a g e n t ,  ( K i r c h and B e r g e i m ,  an i s o a m y l a l c o h o l s o l u b l e ,  a quantitative 1943)  b a s e d upon  p i n k t o r e d compound  by r e a c t i n g PABA w i t h d i a z o t i z e d  thiamine.  obtained  The p r o c e d u r e  f o r t h i s method o f d e t e r m i n i n g t h e amount o f f r e e PABA i s given i n Appendix I I I . B r a t t o n and M a r s h a l l ' s s u l p h o n a m i d e 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 u s e d  fora  - I l l -  quantitative determination and  i n blood  Ekert  o f PABA i n y e a s t  ( E c k e r t , 1943).  (1943) i s e x t r e m e l y  1941)  (Bl nchard, a  This r e a c t i o n d e s c r i b e d by  sensitive  a n d one m i c r o g r a m o f  PABA c a n r e a d i l y be d e t e r m i n e d i n t e n m l . o f 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 o f volumes a still it  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 , a n d s i m i l a r l y  w o u l d be p o s s i b l e t o d e t e r m i n e 0.1 m i c r o g r a m i n one m l .  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) h a v e r e c e n t l y p u b l i s h e d methods b y w h i c h PABA may be d e t e r m i n e d i n o r g a n i c From t h e a b o v e r e v i e w  fluids. o f t h e methods u s e d t o d e t e r -  mine PABA a n d f r o m T a b l e 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 c a n d e t e c t much s m a l l e r amounts o f PABA than  can the present  chemical  tests.  - 112 -  TABLE B. M i c r o b i o l o g i c a l Methods f o r t h e Q u a n t i t a t i v e A s s a y o f PABA  Microorganism Employed  Methods u s e d to record levels of PABA  Clostridium ac e t o b u t y l i cum  Turbidity of culture  0.00003 0.002  Lampen and Peterson  L.  Acid Production  0.0002 0.005  Lewis(l942)  Turbidity of culture  0.001 0.003  Landy and  arabinosus  Acetobacter suboxydans  S m a l l e s t Quant i t y Detectable (micrograms p e r m l . medium)  Reference  (1944)  Dicken(l942)  L a n d y and Streightoff  (1943)  Neurospora crassa  Area of growth  0.0003 0.001  Thompson e t  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 a n d C h e m i c a l Aminobenzoic Acid  Assays  of p_-  The methods g i v e n b e l o w a r e e s s e n t i a l l y v e r b a t u m accounts A.  from t h e o r i g i n a l p u b l i c a t i o n s .  Microbiological  Methods;  (a) I n 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 s s a y f o r t h e d e t e r m i n a t i o n o f PABA o u t l i n e d by L a n d y a n d D i c k e n (1942) was c h o s e n  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  procedure  g i v e n f o r s u c h an a s s a y i s as f o l l o w s : Organism: A.T.C. 621, (0.5  The t e s t o r g a n i s m  a n d i s c a r r i e d on y e a s t  per cent Bacto-yeast e x t r a c t ,  per cent Bacto-agar, at monthly Inoculum  pH 6.0).  e m p l o y e d i s A.  suboxydans.  extract-glycerol-agar 5 per cent g l y c e r o l ,  Stock cultures  1.5  are transferred  i n t e r v a l s and a r e r e f r i g e r a t e d i n t h e i n t e r i m .  f o r assay i s p r e p a r e d by t r a n s f e r f r o m t h e s t o c k  c u l t u r e t o a f l a s k o f b a s a l medium ( T a b l e C) t o w h i c h i s added 0.05 culture  micrograms p-aminobenzoic  i s i n c u b a t e d a t 30°C. f o r 24 B a s a l Medium;  g i v e n i n T a b l e C. centration.  acid.  The i n o c u l u m  hours  p r i o r to use.  The c o m p o s i t i o n of t h e b a s a l medium i s  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-  - 114  Assay Procedure:  -  F i v e c c . o f b a s a l medium a n d 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 t o 5 c c . w i t h water are p l a c e d  i n 50  c c . Erlenmeyer f l a s k s .  m i c r o g r a m s o f PABA.  cluded. a t 15  Blanks  Similarly, a  c o n t a i n i n g f r o m 0.01.  set of r e f e r e n c e f l a s k s i s prepared 0.1  distilled  to  c o n t a i n i n g no PABA a r e i n -  The f l a s k s a r e p l u g g e d w i t h c o t t o n a n d a u t o c l a v e d f o r 15 m i n u t e s .  pounds p r e s s u r e  inoculated with a suspension  A f t e r c o o l i n g they are  o f k. s u b o x y d a n s .  A 24  hour  c u l t u r e grown a s 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  saline solution.  c c . of s t e r i l e  a r e r e s u s p e n d e d i n 15 (approximately serves 48  0.05  cc. of saline solution.  c c . ) of t h e r e s u l t i n g  as t h e i n o c u l u m .  each f l a s k t o d i l u t e  suspension  per f l a s k  a t 30°C. f o r  c c . of w a t e r a r e added t o  t h e c u l t u r e s u i t a b l y f o r measurement o f  t u r b i d i t y and the contents The  cells  One d r o p  The f l a s k s a r e i n c u b a t e d  F o l l o w i n g i n c u b a t i o n , 10  hours.  The w a s h e d  are thoroughly  growth response t o increments  m i x e d by s h a k i n g .  of PABA i s d e t e r m i n e d b y  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 . standard  curve  c a n b e c o n s t r u c t e d by p l o t t i n g  readings  a g a i n s t the c o n c e n t r a t i o n  of m a t e r i a l under t e s t  o f PABA.  i s then read  the colorimeter The PABA  from t h e standard  ( b ) P a r k and Wood (1942) i l l u s t r a t e d a t i v e m i c r o b i o l o g i c a l assay butylicum,  an a n a e r o b i c  content curve.  semi-quantita-  Clostridium aceto-  b a c i l l u s , was u s e d a s i t grows  luxuriantly i n a relatively certain specific  f o r PABA.  simple  A  s y n t h e t i c medium  provided  g r o w t h f a c t o r s a r e a d d e d . " The p r o c e d u r e  - 115 -  TABLE C. B a s a l Medium f o r PABA A s s a y  (Undiluted).  0.6 gm. 10.0 it 20.0 mgm. it 15.0 M 100.0 100.0 It It 40.0 It 2.0 II. 2.0 II 2.0 200.0 m i c r o g m s . it 200.0  Casein hydrolysate Glycerol Tryptophane Cystine K HP0^ KH P0 MgS0 .7H 0 NaCl FeSO, ,7H 0 MnS0 .2H 0 Calcium pantothenate Nicotinic Acid D i s t i l l e d w a t e r t o pH a d j u s t e d 6.0 + 1 2  2  4  4  2  4  2  2  100.0 cc .  o u t l i n e d by these i n v e s t i g a t o r s B a s a l Medium:  i s as f o l l o w s :  The b a s a l medium u s e d h e r e was t h a t  d e s c r i b e d by Oxford and h i s co-workers  (1940) a n d h a d t h e  following composition: Glucose Asparagin ( o r (NH ) HP0 ) K HP0 KH P0 MgS0 .?H 0 NaCl FeS0 .7H 0 MnCl .4H 0 Reduced F e . D i s t i l l e d water 4  2  4  2  4  4  2  4  2  2  2  2  4  20.0 gms. 1.0 0.5 " 0.5 " 0.2 " 0.01 « 0.01 " 0.01 » 3-5 mgms. 1000 c c .  The medium was t u b e d 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 a n d was s t e r i l i z e d by s t e a m i n g f o r 20  - 116  minutes just before to  use.  Prior to sterilization  supplements  t h e b a s a l medium were a d d e d . C u l t u r a l Methods;  butylicum  c u l t u r e s of £ 1 .  Stock  were grown i n b a s a l medium e n r i c h e d  Under t h e s e  c e n t r i f u g a l i z ed,  the suspension  the supernatant  b a s a l medium.  was u s e d as a s t a n d a r d  found t h a t t h e simple  being  0.5  inoculum.  anaerobic  c c . of Following  anaerobic a l l y .  o a t - j a r method ( C a r r o l and  i n which standard  moistened oats  producing  d i s c a r d e d , and t h e organisms  i n o c u l a t i o n , t h e t u b e s were i n c u b a t e d  production  peptone.  c c . of t h e p e p t o n e c u l t u r e was  r e s u s p e n d e d i n 5 c c . of s t e r i l e  before  with  Growth was f o u n d t o .be maximum i n  h o u r s , a t w h i c h t i m e 10  1925)  -aceto-  c o n d i t i o n s t h e organ-ism grox^s r a p i d l y ,  l a r g e amounts o f g a s . 48  -  I t was  Hastings,  c o n t a i n i n g 3 inches of  jars  a t t h e bottom, s e a l e d and e v a c u a t e d by s u c t i o n  placed  i n t h e 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 t h e  of anaerobiosis.  Supplement s t o B a s a l Medium:  Crystalline biotin  d i s s o l v e d i n t h e b a s a l medium t o make f i n a l  concentration i n  t h e c u l t u r e t u b e s f r o m 1.5  x 10"^  x 10~& m i c r o g r a m s p e r c c .  PABA was a d d e d t o t h e b a s a l  similarly,  Estimation  6 m i c r o g r a m s p e r c c . t o 1 x 10"  of B a c t e r i a l Growth:  medium a p p e a r s f i r s t When gas p r o d u c t i o n  medium  i n the c u l t u r e tubes  9 r a n g i n g f r o m 1 x 10 grams p e r c c .  1.5  micrograms per c c . t o  the f i n a l c o n c e n t r a t i o n s  was  b e t w e e n 48  and 72  micro-  Growth i n t h e b a s a l hours a f t e r i n o c u l a t i o n .  i s noted i n the tubes,  t h e anaerobic  jars  -  are re-evacuated  by  117  suction  -  b e f o r e b e i n g opened.  t h o r o u g h l y shaken t o o b t a i n  an  A 5 c c . s a m p l e of t h e c u l t u r e chamber o f a p h o t o e l e c t r i c  B.  Chemical The  even s u s p e n s i o n o f i s then  colorimeter  pipetted and  procedure  o b t a i n e d by r e a c t i n g ( K i r c h and  into  the  readings  are  0.3 pH  water  c c . o f 35  PABA w i t h  soluble  diazotized  thiamine  the  t o a b o u t 11.6,  shift  over tures  followed  amyl a l c o h o l ,  anhydrous sodium s u l f a t e .  The under t h e  5cc.  0.5  read i n a  smallest  The  cc.  resulting portion  of a  e q u a l v o l u m e s of chloride  with  the s o l u b i l i t y of the  i s t h o r o u g h l y shaken, a l c o h o l  i s then  A  i s added.  a c i d i s added, making the f i n a l  mixture  tested  the  5 c c . of  c c . of 35  pH  isoamyl  compound i n per  cent  a b o u t 5.3.  The  separated,  series  cent  Then 4 c c . o f  colored  pH  0.2  and 2 per  NaOH s o l u t i o n i s a d d e d , b r i n g i n g  d i r e c t i o n of t h e  acetic  of thiamine  of sodium n i t r i t e ,  approximately 1 N  To  i s added; the  i s made by m i x i n g  aqueous s o l u t i o n  alcohol.  acid  s h o u l d be a b o u t 2.9.  diazo s o l u t i o n which  of t h e m i x t u r e  i s as  Bergeim, 1943):  per c e n t a c e t i c  aqueous s o l u t i o n  PABA,  p i n k t o r e d compound  i s added t o make a v o l u m e o f a b o u t 20  of t h e m i x t u r e  p e r cent  taken.  f o r d e t e r m i n i n g t h e amount o f f r e e  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 distilled  organisms.  Methods;  b a s e d - u p o n an i s o a m y l a l c o h o l  follows  Each tube i s  and  of c o l o r e d  dried mix-  spectrophotometer.  amount o f PABA f o u n d t o g i v e a  above c o n d i t i o n s  was  a b o u t 10 m i c r o g r a m s .  color This  - 118 -  determination sensitive  shows t h a t  as some m i c r o - o r g a n i s m s  1 x 10~^ m i c r o g r a m s Methods u s e d and  t h e 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  per c c . i n t h e i r growth  medium.  t o d e t e r m i n e t h e amount o f PABA i n u r i n e  also the determination  by K i r c h and B e r g e i m  c a n d e t e c t a s l o w as  o f c o n j u g a t e d PABA a r e o u t l i n e d  (1943).  - 119 -  APPENDIX I V . C o m p o s i t i o n o f M e d i a Used i n t h e P r e v i o u s E x p e r i m e n t s• I.  H o l d i n g Medium f o r A. s u b o x y d a n s .  ( U n d e r k o f l e r and Fulmer, 1937)  0.5 Bacto y e a s t - e x t r a c t Glycerol ( C P . ) 5.0 Bacto-agar 1.5 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 m i n u t e s a t pressure II.  15 p o u n d s  C u l t u r e Medium u s e d 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 Glycerol ( C P . ) 5.0 Bacto-agar 1.5 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 m i n u t e s a t pressure.  III.  p e r cent per cent p e r Gent  Fortified  per c e n t p e r cent per cent 15  pounds  B a s a l Medium ( u n d i l u t e d ) f o r PABA ( L a n d y and S t r e i g h t o f f , 1943)  Casein hydrolysate 0.6 gms. 10.0 ti Glycerol Tryptophane 20.0 mgm. Cystine 15 .0 it Adenine 1.0 ti Quanine 1.0 i i Xanthine 1.0 i i it 100.0 K HP0 II 100.0 KH P0, MgSO, ,7H 0 40.0 ti 2.0 it FeS0?.7H 0 MnSO^,4H 0 2.0 it NaCl 2.0 I I Calcium Pantothenate 200.0 "micro it Nicotinic Acid 100.0 D i s t i l l e d water t o 100.0 m l . pH a d j u s t e d t o 6.0 t 0,1 S t e r i l i z e d f o r 15 m i n u t e s a t 15 pounds pressure. 2  4  2  2  2  2  Assay  - 120 -  APPENDIX  V.  D e t e r m i n a t i o n o f t h e Number of B a c t e r i a l C e l l s i n the Inoculum.  The d r o p p l a t e t e c h n i q u e of M i l e s a n d M i s r a to  e s t i m a t e t h e number  c e r t a i n volume o f c e l l  of b a c t e r i a l c e l l s  (1938)  present i n a  s u s p e n s i o n was e m p l o y e d .  Methodr D r o p p i n g p i p e t t e s were made.  They were  by means o f w e i g h i n g a c c u r a t e l y a known distilled  w a t e r e j e c t e d f r o m them.  f i v e weighings of t h i s cubic  c e n t i m e t e r was Eight f o l d  cell  number  t y p e , t h e a c t u a l number  of drops per  pipette.  d i l u t i o n s were u s u a l l y made, 1.0 c c . o f  s u s p e n s i o n added t o 9.0  Glycerol-yeast  cc. of diluent,  and a f r e s h  dilution. 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 h o u r s a t room t e m p e r a t u r e . sufficiently  of drops of  From t h e a v e r a g e o f  c a l c u l a t e d f o r each  p i p e t t e used f o r each  was  calibrated  d r i e d , f i v e w e l l spaced drops  When t h e a g a r of t h e  d i l u t i o n s o f 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 t h e a b s o r p t i o n of t h e drops  (usually  requires  20-30 m i n u t e s ) , t h e 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  a t 30°C. C o u n t s w e r e made i n t h e d r o p 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 o f c o n f l u e n c e o r o f  - 121 -  g r o s s 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  counts. From t h e mean f i g u r e ,  t h e number o f m i c r o b i a l  cells  p e r c c . o f s u s p e n s i o n was c a l c u l a t e d by t h e f o l l o w i n g formula: C x N x D  =  Number  of c e l l s  per cc. of suspension,  where C i s t h e a v e r a g e number o f c o l o n i e s c o u n t e d itial  d r o p ; N, t h e p i p e t t e c a l i b r a t i o n ,  per c c . d i s t i l l e d water; original  suspension.  per i n -  i e . number o f d r o p s  a n d D, t h e d i l u t i o n  of the  - 122 -  APPENDIX V I . The G e n e r a l E l e c t r i c  M e r c u r y V a p o r , 15 w a t t G e r m i c i d a l  Lamp.  The d e s c r i p t i o n o f t h e lamp u s e d f o r t h e s o u r c e o f ultraviolet follows  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  (General E l e c t r i c  B u l l e t i n , 1947):  Designat i o n  ^  Nominal Length, inches Diameter, inches Bulb A p p r o x i m a t e Lamp Amperes A p p r o x i m a t e Lamp V o l t s  18 1 T-8 0.31 55  C i r c u i t Voltage Rated Average L i f e , hours Base U l t r a v i o l e t Output,  110-125  2500  Med.  Bipin  w a t t s a t 2537^ a t  List  100 h o u r s p r i c e , each  This General E l e c t r i c Lamp, a s s e e n f r o m t h e ' t a b l e o its  2.9  #4.50 M e r c u r y V a p o r 15 w a t t  Germicidal  g i v e n b e l o w r a d i a t e s most o f  e n e r g y at t h e 2537A l i n e w h i c h i s v e r y n e a r t h e wave  l e n g t h most e f f e c t i v e  Irradia tion  i n destroying  bacteria.  Procedure :  A known amount o f a s o l u t i o n up t o 20 c c . i s m e a s u r e d into a petri and 1.0  d i s h h a v i n g t h e d i m e n s i o n s : 9.0  cm. i n d i a m e t e r  cm. d e e p . The s o l u t i o n i s t h e n i r r a d i a t e d by t h e p r e v i o u s l y  I  - 123 -  described The  14.0  ultraviolet  source f o r various  time  d i s t a n c e f r o m t h e lamp t o t h e s u r f a c e  intervals.  of t h e s o l u t i o n i s  cm. At t h e end o f t h e i r r a d i a t i o n ,  f u l l y made up t o i t s o r i g i n a l  the solution i s care-  volume w i t h t h e d i l u e n t .  a p p a r e n t l o s s i s due t o t h e e v a p o r a t i o n  The  of t h e d i l u e n t by  the heat o f t h e lamp. TABLE D. 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