Studies on Growth and Metabolism in Colipidium Campylum

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FORDHAM UNIVERSITY GRADUATE SCHOOL

.............................. 1950

This dissertation prepared under my direction by

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entitled

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STUDIES ON GROWTH AID METABOLISM

I I CQLPIDIUM CAMFYLUM

has been accepted in partial fulfilment of the requirements for the

% Degree of

Dr. C h a rle s W ilb er (Faculty Adviser)

STUDIES ON GROWTH AND METABOLISM IN COLPIDIUM CAMPYLUM

BY BENEDICT B. O'MALLEY A.B*, Fordham U n iv e r s ity , '4 4 M .S ., Fordham U n iv e r s ity , '4 8

DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF BIOLOGY AT FORDHAM UNIVERSITY NEW YORK 1950

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ProQuest Number: 10992968

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 10992968 Published by ProQuest LLC(2018). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

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TABLE OF CONTENTS Page LIST OF FIGURES . . . . . ........... . . . ............ ................... ACKNOWLEDGMENTS I* II*

.................

INTRODUCTION

v ii

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MATERIAL AND METHODS

......... . ...........................

I n o r g a n ic s e r i e s .......................... S u cro se s e r i e s ............................ V itam in s e r i e s ............................... Hormone s e r i e s • • • • • • • • . .......................... G en eral rem arks ............... III.

IV*

V*

OBSERVATIONS ANDRESULTS

1 6 6 8

10 11 14

...............................

15

G en eral ••••• S u c ro se s e r i e s ••••• I n o r g a n ic s e r i e s ........... •••• V itam in s e r i e s ............ Optimum c o n c e n tr a tio n s tu d i e s ............... ........................ R ib o f la v in s e r i e s P a n to th e n ic a c id s e r i e s ••••••••••••.. Hormone s e r i e s

15 16 16 20 21 23 25 28

DISCUSSION

..........................

77

G en e ral ........... S u c ro se s e r i e s ....................... ..................... I n o r g a n ic s e r i e s V itam in s e r i e s ............. ••••• ............................... • • • • • • Hormone s e r i e s

77 77 81 84 89

SUMMARY

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BIBLIOGRAPHY

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LIST OF FIGURES F ig u re 1.

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Page Fhotom i c ro g ra p h o f C olpidium campylum i n t h e c o n t r o l s e r ie s * M a g n ific a tio n X ......

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P h o to m icro g rap h o f C olpidium campylum i n t h e c o n t r o l s e r i e s s ta in e d w ith Sudan b la c k . M a g n if ic a tio n X4 5 0 * . . . . . . .

55

P h o to m icro g rap h o f C olpidium campylum i n t h e in o r g a n ic s e r i e s . M a g n if ic a tio n X 430 ......................................................

57

P h o to m ic ro g rap h o f C olpidium campylum i n t h e s u c ro s e s e r i e s . M a g n ific a tio n X 430 ..................................................... P h o to m icro g rap h o f C olpidium campylum i n th e c o n t r o l s e r i e s . M a g n ific a tio n X120

57 39

P h o to m icro g rap h o f C olpidium campylum i n t h e s u c ro s e s e r i e s . M a g n ific a tio n X 120 ..................................................

59

P h o to m icro g rap h o f C olpidium campylum i n t h e s u c ro s e s e r i e s a f t e r b ein g s ta i n e d w ith Sudan b lack * M a g n ific a ­ tio n X 120 .......................................... P h o to m icro g rap h o f C olpidium campylum i n th e f i r s t h a l f o f th e grow th s tu d ie s i n t h e p r o g e s te r o n e s e r i e s . M ag n ifi­ c a t i o n X 120 ..................................................... P h o to m ic ro g rap h o f C olpidium campylum i n t h e f i r s t h a l f o f th e grow th s tu d ie s i n th e t e s t o s t e r o n e s e r i e s . M ag n ifi­ c a t i o n X 120 P h o to m icro g rap h o f C olpidium campylum i n t h e f i r s t h a l f o f th e grow th s tu d ie s i n th e e s t r a d i o l s e r i e s . M ag n ifica­ t i o n X 100 ...................................................

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F ig u re 11*

1 2

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15.

14.

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16. 17. 18. 19. 20.

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Page P h o to m icro g rap h o f C abldium campylum i n t h e seco nd h a l f o f th e grow th s tu d ie s i n th e te s to s te ro n e s e r i e s . M a g n if ic a tio n X 1 2 0 ...............................

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P h o to m icro g rap h o f C olpidium campylum i n t h e seco nd h a l f o f th e grow th s t u d i e s i n th e te s te r o n e s e r i e s . M a g n if ic a tio n X 120 . . • » • • . . • • . « ...............

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P h o to m icro g rap h o f C olpidium campylum i n th e second h a l f o f t h e grow th s tu d i e s i n th e p ro g e s te r o n e s e r i e s . ........... M a g n if ic a tio n X 120

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P h o to m icro g rap h o f C olpidium campylum i n th e second h a l f o f th e grow th s t u d i e s i n th e e s t r a d i o l s e r i e s . Magni­ f i c a t i o n X 1 2 0 ...........

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Graph showing t h e grow th o f C olpidium campylum i n th e optimum c o n c e n tr a tio n s o f th e v ita m in s e r i e s ...............

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Graph show ing t h e grow th o f C olpidium campylum i n th e hormone s e r i e s

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Graph showing th e grow th o f C olpidium campylum i n v a r io u s m edia ...............

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Graph show ing t h e grow th o f C olpidium campylum i n v a r io u s m edia

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Graph show ing t h e grow th o f C olpidium campylum i n v a r io u s m edia . . . . . . . . . . . . . . . . .

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G raph i l l u s t r a t i n g th e m o rp h o lo g ic a l changes t h a t ta k e p la c e d u rin g th e a d a p t­ a t i o n p ro c e s s i n th e in o r g a n ic and s u c ro s e m edia

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Graph i l l u s t r a t i n g th e m o rp h o lo g ic a l changes t h a t accompany th e grow th p ro ­ c e s s i n th e hormone s e r i e s ...........

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F ig u re 22.

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Page Graph i l l u s t r a t i n g th e grow th o f C olpidium campylum i n th e th ia m in e h y d r o c h lo rid e s e r i e s ....................

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Graph i l l u s t r a t i n g th e grow th o f C olpidium campylum i n th e r i b o f l a v i n s e rie s

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Graph i l l u s t r a t i n g th e g row th o f C olpidium campylum i n t h e p a n to th e n ic a c id s e r i e s

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( l ) Scheme r e p r e s e n tin g th e p o s s ib le m e ta b o lic pathw ays o f th e grow th p ro ­ c e s s i n t h e s u c ro s e s e r i e s ......... (2) Scheme r e p r e s e n tin g th e p o s s ib le m e ta b o lic pathw ays o f th e grow th p ro ­ c e s s i n th e a c e t a t e medium • • • • . • • . « (5) Scheme r e p r e s e n tin g th e i n t e g r a ­ t i o n o f p r o t e i n , f a t and c a rb o h y d ra te m etab o lism

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Scheme r e p r e s e n tin g th e r o l e p la y e d i n th e m etab o lism o f C olpidium campylum by th e v a r io u s v ita m in s . . .

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Scheme r e p r e s e n tin g th e p o s s ib le m e ta b o lic pathw ays o f th e grow th p r o c e s s i n th e hormone s e r i e s . . . . • •

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I l l u s t r a t e s th e p e rc e n ta g e o f p r o te o u s p e p to n e i n th e in o r g a n ic and s u c r o s e s o lu tio n s d u rin g th e te n tra n s fe rs ...........

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ACKNOWLEDGMENTS

The a u th o r i s in d e b te d t o D octor C h a rle s G. W ilb e r who su g g e ste d t h i s problem , and u n d e r whose d i r e c t i o n th e i n v e s t i g a t i o n was c a r r i e d on*

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STUDIES ON GROWTH AND METABOLISM IN COLPTDIUM CAMPYLUM.

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I.

INTRODUCTION

I t i s w e ll known t h a t p l a n t s and th o s e org an ism s c o n ta in ­ in g c h lo r o p h y ll a r e a b l e t o s y n th e s is e complex o r g a n ic compounds from in o r g a n ic s a l t s and w ater* I n 1887 V ino g rad sk y b ro u g h t t o l i g h t th e c h e m o sy n th e tic p r o p e r t i e s o f c h lo r o p h y ll- la c k in g b a c te r ia *

The c o l o r l e s s

f l a g e l l a t e Chilom onas Param ecium , i s a n o th e r a p p a re n t e x c e p tio n t o th e g e n e r a l r u l e t h a t l i v i n g o rg an ism s la c k in g c h lo r o p h y ll m ust r e s o r t to t h e u t i l i z a t i o n o f o r g a n ic compounds t o m a in ta in l i f e (M ast and P a c e , 1 9 3 3 ).

O ltsm anns d e s c rib e d Chilom onas

Param ecium a s an o rg an ism c o n ta in in g no chrom atophores and con­ s e q u e n tly d ep en d en t on o r g a n ic compounds f o r l i f e *

Both

O ltm anns (1922) and P rin g sh e im (1921) came t o th e c o n c lu s io n t h a t Chilom onas Param ecium i s a s a p ro p h y tic o rg an ism .

P rin g sh eim

f u r t h e r s t a t e d t h a t th e l e a s t com plex s o u rc e s o f n itr o g e n and c arb o n n e c e s s a r y f o r g ro w th a r e g ly c in e and sodium a c e t a t e r e ­ s p e c tiv e ly * M ast and P ace (1933) r e f u t e d th e above when th e y w ere a b le t o c u l t u r e t h i s o rg an ism on a medium t h a t c o n ta in e d o n ly t h r e e s a l t s , Mg4 S0 g,K 2 HP0 4 , and NH4 C I.

The c u l t u r e s w ere found t o

f l o u r i s h p r o v id in g t h e CO gcontent o f t h e a i r was in c r e a s e d and s i l i c o n added t o t h e s o l u t i o n .

T h ere was no complex o r g a n ic

compound su ch a s g ly c in e p r e s e n t , w hich P rin g sh e im m a in ta in e d

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m ust be p r e s e n t f o r l i f e f u n c tio n s t o ta k e p la c e * T h is a b i l i t y o f an o rganism t o a d ap t i t s b io c h e m ic a l p ro ­ c e s s e s so a s t o s u s t a i n l i f e i n a sim p le environm ent p o se s s e v e r a l q u e s tio n s *

o n e , w h e th e r t h i s a b i l i t y i s l i m i t e d o r wide­

s p re a d i n n a tu r e and tw o , how sim p le th e medium can become o r how f a r t h e i n d i v i d u a l o rganism can go i n i t s b io c h e m ic a l modi­ f i c a t i o n s so a s t o m a in ta in l i f e and a llo w t h e organism t o r e ­ p ro d u c e , r e s p i r e and p erfo rm t h e f u n c tio n s c h a r a c t e r i s t i c o f l i v i n g organism s* E x p e rim en ts w ere co n d u cted i n an a tte m p t t o answ er t h i s q u e s tio n by s e l e c t i n g an organism more complex i n n a tu r e and s t r u c t u r a l d i f f e r e n t i a t i o n th a n f l a g e l l a t e s Chilom onas param ecium , w hich b elo n g s t o t h e m ost p r im i t i v e c l a s s o f t h e P ro to zo a* o rg an ism s e le c t e d was t h e c i l i a t e C olpidium campylum*

The

T h is o rg an ­

ism h a s been u s e d e x te n s iv e ly i n b io -c h e m ic a l and c e l l u l a r m eta­ bolism * M ast and P a c e (1 9 5 8 , 1946) and P ace (1 9 4 7 ), to o k ad v an tag e o f t h e sim p le medium a s an a lm o st i d e a l s i t u a t i o n i n w hich t o s tu d y th e phenomenon o f a l l e l o c a t a l y s i s , and s tu d i e s on th e na­ t u r e o f t h e g row th p ro m o tin g s u b s ta n c e i n Chilom onas Paramecium i n d i c a t e d t h a t i n low c o n c e n tr a tio n s i t s tim u la te s and i n h ig h c o n c e n tr a tio n s i t i n h i b i t s grow th*

S p e c u la tio n a r o s e a s t o

w h eth er i t was a s i n g l e s u b s ta n c e o r w h eth er i t was a co m b in atio n o f two s u b s ta n c e s one w hich s tim u la te d and one w hich i n h i b i t e d th e grow th p r o c e s s . L

P ac e (1944) s t a t e d f u r t h e r t h a t i f t h e r e

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a r e two s u b s ta n c e s th e y a r e so much a l i k e i n chem ical s t r u c t ­ u r e and c h a r a c t e r i s t i c s t h a t th e y can n o t be s e p a r a te d .

A ll a t ­

te m p ts to i s o l a t e t h e two s u b s ta n c e s have been u n s u c c e s s f u l. P ac e (1947) i n v e s t i g a t e d th e p o s s i b i l i t y t h a t th e grow th prom ot­ in g s u b s ta n c e s m ig h t be one o f th e B complex group o f v ita m in s . H is e x p e rim e n ts i n d i c a t e t h a t i n v e ry l a r g e c o n c e n tr a tio n s th e g ro w th o f Chilom onas param ecium was in c r e a s e d , w ith t h e a d d i­ t i o n o f t h e B group o f v ita m in s . The work o f R o b e rtso n (1 9 2 4 ), M arbarger (1 9 4 3 ), and K id d e r (1959) w ith C olpidium c o n firm s t h e p re s e n c e o f a grow th prom ot­ in g s u b s ta n c e i n t h e C i l i a t a .

The q u e s tio n th e n a r i s e s a s to

t h e n a t u r e o f t h i s gro w th prom oting s u b s ta n c e i n t h e C o lp id ia . The work o f P ace (1947) on Chilom onas param ecium

w ith t h e B

group o f v ita m in s was r e p e a te d w ith C olpidium campylum i n t h i s s e t o f e x p e rim e n ts .

The a c t i o n o f A sc o rb ic A cid, Fe C lg , and

C h o lin e c h lo r id e on t h e g ro w th o f C olpidium campylum was a l s o in v e s tig a te d .

A n o th er g ro u p o f p o w erfu l c e l l u l a r s tim u la n ts

th e horm ones w ere s e l e c t e d a s a p o s s i b le grow th p ro m o tin g f a c t o r and t h e i r e f f e c t on th e grow th o f C olpidium campylum was s t u d i e d . n ite reaso n s.

The s t e r o l hormones were s e le c te d f o r two d e f i ­ F i r s t l y b e c a u se t h e s t e r o l s f o r some tim e have

been u s e d a s c a rc in o g e n s i n e x p e rim e n ts co n cern ed w ith abnorm al c e llu la r p o life r a tiv e s tu d ie s .

The b a s ic s t e r o l s u b s ta n c e

c h o l e s t e r o l , was s tu d ie d a s a p o s s ib le grow th p ro m o tin g sub­ s ta n c e by S u lliv a n (1949) and i t was found t h a t c h o l e s t e r o l L

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p ro d u c ed s tin m la tin g e f f e c t s on t h e grow th o n ly a f t e r th e o r ­ ganism s became acclim ated t o t h e medium*

S eco n d ly , no work h as

b een done a t a l l on th e e f f e c t o f th e s t e r o l hormones w ith P ro to z o a *

The o n ly work done on th e e f f e c t o f th e hormones

and grow th s ti m u l a t i o n i n th e P ro to z o a in v o lv e d t h e u s e o f T h y ro id E x tr a c ts and T hyroxine*

The r e s u l t s o f t h e work w ere

h ig h ly c o n t r o v e r s i a l and no c l e a r c o n c lu s io n s were reached* Some i n v e s t i g a t o r s su ch a s N ow ikoff (1908) w ith t h e C i l i a t a , Shumway (1 9 1 4 , 1929) w ith Param ecium , B uddington and Harway (1915) w ith t h e C i l i a t a and A bderhalden and S ch iffm an (1922) w ith Param ecium , o b ta in e d p o s i t i v e grow th in c r e a s e w ith th e a d d i t i o n o f t h e T h y ro id hormone*

A g r e a t d i f f e r e n c e i n th e

r e s u l t s was o b ta in e d when t h e i n v e s t i g a t o r s u se d cru d e e x t r a c t s o f t h y r o id and p u re

th y ro x in e *

The work o f W ertheim er (1927)

and Capua (1951) added t o th e g e n e r a l s t a t e o f c o n fu sio n when t h e i r e x p e rim e n ts d is c lo s e d no p o s i t i v e e f f e c t o f any s o r t on t h e grow th o f I n f u s o r i a by t h e a d d it io n o f th y ro x in e *

I t is

i n t e r e s t i n g t o n o te t h a t th o s e a u th o r s who have n o tic e d an i n ­ c r e a s e d r a t e o f f i s s i o n fo llo w in g t h e a d d it io n o f t h y r o id p r e ­ p a r a t i o n s have a l s o o b se rv e d a s i z e d e c re a s e i n t h e organism s* N ow ikoff (1908) e x p la in e d t h i s s i z e d if f e r e n c e a s th e s p e c i f i c r e s u l t o f t h e t h y r o i d p r e p a ra tio n *

Some c o n fu s io n e x i s t s a l s o

a s t o changes i n p h y s io lo g y o f th e b e a s ts u n d e r th e i n f lu e n c e o f th e hormone* The in f o r m a tio n t h a t i s a v a i l a b l e i n r a t h e r m eager on L

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n s e v e r a l c o u n ts f i r s t l y i n view o f t h e g r e a t number o f hormones i n e x is te n c e and th e f a c t t h a t o n ly one o f a v a s t number o f a v a i l a b l e hormones h a s been s tu d ie d a s a p o s s ib le grow th p ro ­ m o tin g s u b s ta n c e , and s e c o n d ly t h e in fo rm a tio n t h a t i s a v a i l ­ a b le on t h e t h y r o id group o f s u b s ta n c e s h as n o t been to o r i g i d ­ l y c o n t r o ll e d n o r h a s i t been done u n d e r th e c o n d itio n s o f s t e r i l ­ i t y o f th e medium, t h u s , th e f a c t o r o f b a c t e r i a and o t h e r o rg an ­ ism s b ein g p r e s e n t h a s o b sc u re d w h atev er c l e a r p o i n t s t h a t may h ave been co n clu d ed from th e s tu d i e s th u s f a r* I t w as, t h e r e f o r e , d e c id e d t o stu d y th e s t e r o l group o f horm ones and t h e i r e f f e c t on th e g row th o f C olpidium campylum u n d e r t h e c o n t r o ll e d c o n d itio n s o f p u re s t e r i l e c u ltu r e s *

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II.

MATERIAL AND METHODS

S t e r i l e c u l t u r e s o f C olpidium campylum w ere o b ta in e d from o u r L a b o r a to r ie s i n f a t - f r e e p r o te o s e - p e p to n e media* The m edia i n w hich t h e o rg an ism s w ere grow ing was t e s t e d f o r t h e p re s e n c e o f b a c t e r i a by s tr e a k in g a g a r p l a t e s e v e ry 5 days and in c u b a tin g a t 37 d e g re e s C*

A d u p l ic a te s e r i e s o f

p l a t e s was a l s o in c u b a te d a s c o n tro ls *

I n t h i s way t h e s t e r ­

i l i t y o f t h e medium was c o n t in u a lly t e s t e d f o r any p o s s i b le co n tam in atio n * The p o p u la tio n o f th e c u l t u r e s was e s tim a te d by d i l u t ­ in g a sm a ll w eighed d ro p o f th e medium w ith w a te r to make cc*

10

One d ro p o f t h i s d i l u t i o n was p la c e d on a s l i d e ; th e

s l i d e p a s s e d g e n tly o v e r a fla m e t o im m o b ilize th e o rganism s and th e c o u n t was made* in

1

cc.

By a s c e r t a i n i n g th e number o f d ro p s

o f w a te r and th e number o f d ro p s i n

1

c c . o f medium

f a c t o r was o b ta in e d w hich when m u l t i p l ie d by t h e number o f o rg a n ism s o n t h e s l i d e r e s u l t e d i n th e number o f org an ism s p e r cc*

A ll t h e c u l t u r e s were grown a t a c o n s ta n t tem pera­

t u r e o f 26*6 d e g re e s C* I n o r g a n ic s e r i e s S t e r i l e c u l t u r e s o f C olpidium campylum a medium which c o n ta in e d th e fo llo w in g s

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were a d a p te d t o

sodium a c e t a t e 150*0

M g., Magne s iu m -s u lp h a te (MgSO^) 1 2 .0 Mg. P o ta ssiu m -h y p o p h o sp h ate (KgHQP^) 2 0 .0 Mg. and Ammonium-chloride (NH^Cl) 5 0 .0 Mg. (Mg. p e r 100 cc o f HgO). The p r o c e s s c o n s is te d o f th e fo llo w in g s t e p s . A s e r i e s o f t e n tu b e s was s e t up i n d u p l ic a te e a c h c o n ta in in g 10 c c . c f t h e above medium*

T h ere was added t o th e f i r s t tu b e

3 c c . o f a f l o u r i s h i n g c u l t u r e o f C olpidium campylum i n 3# p ro ­ t e o s e p e p to n e .

When s u f f i c i e n t tim e had e la p s e d f o r maximum

grow th t o t a k e p la c e ,

2

c c . o f t h e f i r s t tu b e was added by s t e r ­

i l e p i p e t t e s t o t h e second tu b e . o c c u rre d i n t h e second tu b e t o th e t h i r d tu b e .

Once a g a in when maximum grow th

c c . o f th e medium was t r a n s f e r r e d

1

When th e grow th peak had been re a c h e d i n

th e t h i r d t u b e , 5 c c . o f th e medium was t r a n s f e r r e d to th e f o u r t h tube*

The p ro c e s s was r e p e a te d when grow th i n th e f o u r t h

tu b e became maximum and t o th e f i f t h tu b e was added 4 d ro p s o f th e medium o f t h e f o u r t h tu b e . e x a c tly J o f a c c . imum,

2

These f o u r d ro p s r e p r e s e n te d

When grow th i n th e f i f t h tu b e became max­

d ro p s o r e x a c tly

1 /8

o f a c c . o f th e m edia i n th e f i f t h

tu b e was added t o t h e s i x t h tu b e . s i x t h tu b e was re a c h e d , t h e s e v e n th tu b e .

1

When t h e g ro w th peak i n th e

d ro p o r 1 /1 6 o f a c c . was added t o

When grow th was maximum i n t h e s e v e n th tu b e ,

t h e e i g h t tu b e was in n o c u la te d by a w ire lo o p which r e p r e s e n te d 1 /7 0 o f a c c . o f t h e c o n te n ts o f th e s e v e n th tu b e .

The n i n t h

and t e n t h tu b e s w ere in n o c u la te d by th e w ire lo o p te c h n iq u e and t h e s e r i e s o f t e n tu b e s i n d u p l ic a te s e t s w ere a g a in s e t up and a stu d y made o f t h e m o rp h o lo g ic a l changes th ro u g h t e n more t r a n s f e r s i n 2 .5 c c . o f in o r g a n ic medium u s in g t h e w ire lo o p

8 r

-i a s a means o f in n o d ila tio n *

A c o n tr o l o f s e r i e s o f 2 ,5

cc p ro ­

t e o s e p e p to n e i n 10 t e s t tu b e s was s e t up i n d u p lic a te *

The

p e r c e n ta g e o f P r o te o s e P e p to n e , i n th e in o r g a n ic s o lu t i o n i s c a l c u l a t e d i n fig * 28* S u c ro se s e r i e s S t e r i l e c u l t u r e s o f C olpidium campylum i n

fa t-fre e

p r o te o s e p e p to n e medium w ere a d a p te d t o a medium o f *1 M olar S u cro se i n d i s t i l l e d w a te r t y th e fo llo w in g t r a n s f e r s e r i e s * A s e r i e s o f t e n tu b e s w ere s e t up i n t r i p l i c a t e s e ts *

The f i r s t

f i v e c o n ta in e d v a r io u s am ounts o f *1 M* s u c ro s e s o l u t i o n , w h ile t h e l a s t f i v e c o n ta in e d 1 cc* o f *1 M* s u c ro s e each* f i r s t tu b e th e r e was 50 c c . o f

.1

In th e

M s u c ro s e and t o t h i s was

added 5 cc* o f a f lo u r i s h i n g c u l t u r e o f C olpidium campylum i n 5%

f a t - f r e e p r o te o s e p e p to n e medium.

A f te r s u f f i c i e n t tim e

had e la p s e d f o r maximum grow th t o ta k e p la c e , 5 c c . o f th e con­ t e n t s o f th e f i r s t tu b e was added to th e second tu b e w hich con­ t a i n e d 20 cc* o f .1 M su cro se*

A gain, when grow th i n t h e second

tu b e was re a c h e d a peak 4 c c . o f t h e c o n te n ts o f th e second tu b e was added to th e t h i r d tu b e w hich c o n ta in e d

10

c c . o f su cro se*

When grow th i n th e t h i r d tu b e re a c h e d a maximum f i g u r e , 2 c c . o f i t s c o n te n ts was added to th e f o u r th tu b e w hich c o n ta in e d 5 c c . o f .1 m. s u c r o s e . ed i n t h e f o u r t h tu b e ,

When th e grow th peak had been re a c h ­ 2

c c . o f i t s c o n te n ts was added t o th e

f i f t h tu b e w hich c o n ta in e d 2 .5 c c . o f th e s u c ro s e medium* L

_]

When t h e grow th peak i n th e f i f t h tu b e had been re a c h e d , 1 c c , o f i t s c o n te n ts was added to th e s i x t h tu b e w hich c o n ta in e d 1 c c , o f su c ro se *

When maximum grow th i n th e s i x t h tu b e had

b een re a c h e d 1 cc* o f i t s c o n te n t was added to th e s e v e n th tube* T h is p ro c e s s was r e p e a te d u s in g ,5 o f 1 cc* o f th e c o n te n ts o f th e s e v e n th to in n o e u la te th e e ig h th tu b e , and th e same amount from th e e ig h th tu b e t o in n o e u la te th e n in th tu b e*

F iv e d ro p s

o f th e c o n te n ts o f th e n i n th tu b e w ere u se d to in n o e u la te th e t e n t h tube*

A seco n d s e t o f te n tu b e s were th e n s e t up i n t r i p l i ­

c a te which c o n ta in e d 7*5 cc* o f *1 M s u c ro s e s o lu t i o n and th e f i r s t tu b e o f th e s e r i e s was in n o c u la te d by th e w ire lo o p te c h ­ n iq u e*

The w ire lo o p te c h n iq u e was u sed th ro u g h te n s u c c e s s iv e

in n o c u la tio n s i n t h i s s e r i e s and th e grow th p ro c e s s and morpho­ l o g i c a l a d a p ts ,tio n s tu d ie d i n th e p u re s u c ro s e medium*

P e r c e n t­

age o f P r o te o s e P e p to n e i n s u c ro s e s o l u t i o n c a l c u l a t e d i n f i g . 28* Only h ig h p u r i t y c h e m ic a ls , and p y r o g e n - f r e e , p y re x d i s ­ t i l l e d w a te r w ere u se d i n p r e p a r in g t h e c u l t u r e m edia i n th e i n ­ o r g a n ic and s u c ro s e s e r ie s *

S t e r i l e , p y r e x - g la s s p i p e t t e s

w ere u s e d i n th e t r a n s f e r s *

The p i p e t t e s were p re p a re d by p lu g ­

g in g th e o p en ,

Aide end o f each p i p e t t e w ith n o n -a b s o rb e n t c o tto n ,

w rapping t h e b a se o f th e p i p e t t e i n c o tto n and i n s e r t i n g i t i n t o a p y re x t e s t tube*

I n t h i s m anner, th e t i p o f th e p ip ­

e t t e i s su spended i n th e tu b e and th e c o tto n p lu g g in g p r e v e n ts c o n ta m in a tio n due to th e e n tr a n c e o f b a c te r ia *

The p i p e t t e s

w ere th e n s t e r i l i z e d i n th e a u to c la v e a t 20 l b s . p r e s s u r e f o r

10

20 m in u tes*

A p i p e t t e was u s e d o n ly f o r one t r a n s f e r , and was

th e n p u t a s id e f o r r e c le a n in g and s t e r i l i z a t i o n b e f o r e i t was u s e d a g a in .

The m edia was s t e r i l i z e d t y m a in ta in in g th e tem­

p e r a t u r e a t 99 d e g re e s C. f o r 2 hours*

A u to c la v in g o r s t e r i l ­

i z i n g a t h ig h e r te m p e r a tu re s b re a k s down th e c o n s t i t u e n t s o f th e medium and r e n d e r s i t u s e le s s *

The g la s s w a r e , p i p e t t e s ,

and g r a d u a te s u s e d i n c o n n e c tio n w ith th e e x p e rim e n ts were made c h e m ic a lly c le a n t y w ashing i n a s o lu t i o n o f tris o d iu m p h o s p h a te , r i n s i n g 15 tim e s i n t a p w a te r and e i g h t tim e s i n d i s t i l l e d w ater* V itam in s e r i e s P r e lim in a r y o b s e r v a tio n s on t h e B complex g ro u p o f v i t ­ am ins in v o lv e t h e f o llo w in g s u b s ta n c e s , th ia m in h y d ro -c h lo ­ r i d e , r i b o f l a v i n , n i a c i n , p y r id o x in e , p a n to th e n ic a c id and c h o lin e c h lo rid e *

S o lu tio n s o f th e s e compounds w ere made up

i n 2*5 cc* o f p r o te o s e - p e p to n e i n th e o r d e r o f 1%, *1% and .01$ s o lu tio n s *

Those s u b s ta n c e s t h a t p ro d u ced marked p o s i­

t i v e r e s u l t s on th e g ro w th o f C olpidium campylum were f u r ­ t h e r s tu d ie d t o a s c e r t a i n th e e x a c t c o n c e n tr a tio n s t h a t p ro ­ duced maximum grow th*

T hese s t u d i e s e n t a i l e d th e u s e o f

th ia m in h y d r o c h lo rid e , r i b o f l a v i n and p a n to th e n ic a c id and th e optimum c o n c e n tr a tio n , sub-optim um and supra-optim um c o n c e n tr a tio n o f each was a s c e r t a i n e d , i n r e l a t i o n to th e maximum amount o f gro w th p ro d u c e d . L

I n t h i s s e r i e s o f s tu d ie s

11 r

n 12

s e t s o f tu b e s were u s e d f o r each c o n c e n tr a tio n , so t h a t t h i s

s e r i e s i s q u i t e sound s t a t i s t i c a l l y , i n r e g a r d to th e r e s u l t s o f th e g row th s tu d ie s *

B ecause o f h e a t l i a b i l i t y o f t h i s g roup

o f compounds, i t was n e c e s s a r y to fo re g o th e u s u a l te c h n iq u e o f h e a tin g a t 99 d e g re e s f o r

Z

h o u rs and i n s t e a d g r e a t c a r e

had t o be g iv e n t o th e p r e p a r a t i o n o f th e s o lu tio n s i n s t e r i l e p r o te o s e p ep to n e*

I n o r d e r t o i n s u r e th e s t e r i l e c o n d itio n o f

th e s o l u t i o n s one s e t was c o n tin u o u s ly b e in g p l a t e d on a g a r and in c u b a te d a t 57 d e g re e s C* ea c h tim e a new s e r i e s was made up.

A ll s o lu t i o n s i n t h i s s e t were p re p a re d i n an atm o sp h ere

o f u l t r a v i o l e t l i g h t so t h a t no c o n ta m in a tio n was p o s s i b l e . P r e lim in a r y o b s e r v a tio n s were a ls o co n d u cted on th e e f ­ f e c t o f Fe Clg and a s c o r b ic a c i d on t h e grow th o f C olpidium campylum i n

1

%, . 1 % and • 0 1 $ c o n c e n tr a tio n s o f th e s e s u b s ta n c e s

i n 2 .5 c c . o f s t e r i l e p r o te o s e p e p to n e .

S in c e no p o s i t i v e e f ­

f e c t was n o te d no f u r t h e r s t u d i e s on th e optimum c o n c e n tr a tio n o f th e s e s u b s ta n c e s w ere u n d e r ta k e n .

The u s u a l c o n d itio n s o f

s t e r i l i t y w ere o b se rv e d i n t h i s s e t o f e x p e rim e n ts and th e s o lu t i o n s were exposed to 99 d e g re e s C f o r a p e r io d o f 2 h o u rs to in s u re s t e r i l i t y .

I n e a c h o f t h e p r e lim in a r y s e t s o f ex­

p e rim e n ts a c o n tr o l was r u n and p l a t e d r e g u l a r l y on a g a r s l a n t s so a s to i n s u r e th e s t e r i l i t y o f th e e x p e rim e n ts . Hormone s e r i e s The hormones u s e d i n t h i s s e r i e s were o b ta in e d from th e L

12 r

-i S c h e rin g L a b o r a to r ie s i n s t e r i l e am pules o f 1 cc* each and con­ t a i n e d th e r e s p e c t i v e hormones i n a s o lu t io n o f Sesame o il* s e r i e s c o n s is te d o f f o u r p a r ts *

The

From a 1 cc* am pule w hich con­

t a i n e d 25 mg. o f t e s t o s t e r o n e p r o p r io n a te , t h e r e was w ith ­ draw n 3 d ro p s by means o f a c a l i b r a t e d , s t e r i l e p i p e t t e and t h i s added t o a s e r i e s o f 11 tu b e s w hich c o n ta in e d 2*5 c c . o f f a t - f r e e p r o te o s e p e p to n e s o lu tio n * t e s t o s t e r o n e i n p e p to n e was .1 8 5 $ .

The c o n c e n tr a tio n o f

T h ere was added 3 d ro p s o f

sesam e o i l by means o f a s t e r i l e p i p e t t e t o 2 .5 c c . o f p r o te o s e -p e p to n e w hich s e rv e d a s th e c o n tr o l s e r ie s *

One o f th e

t e s t tu b e s o f th e c o n t r o l s e r i e s and th e t e s t o s t e r o n e was p l a t e d on a g a r t o check th e s t e r i l i t y o f th e experim ents*.

The

seco n d p a r t c o n s is te d o f a s e r i e s

w hich was s e t u p a s th e above

b u t c o n ta in e d p r o g e s te r o n e a s th e

hormone t o be s tu d ie d *

a

1

c c . am pule w hich c o n ta in e d

1

From

mg. o f p r o g e s te r o n e t h e r e was

w ith d raw n by means o f a s t e r i l e p i p e t t e 5 d ro p s whose volume was c a l i b r a t e d so a s t o g iv e a c o n c e n tr a tio n o f .148% o f p ro ­ g e s te r o n e when added t o 2 .5 c c . o f p r o te o s e peptone*

L ik e

volum es o f sesam e o i l w ere added t o 3 tu b e s o f 2 .5 c c . p ro ­ t e o s e p e p to n e w hich s e rv e d a s th e

c o n t r o l . One o f th e 11

t e s t tu b e s i n t h e p r o g e s te r o n e s e r i e s and one o f th e tu b e s i n th e c o n t r o l s e r i e s was p l a t e d on a g a r and in c u b a te d a t 57 d e g re e s C. t o check on th e s t e r i l i t y o f t h e e x p e rim e n ts . The t h i r d p a r t o f t h i s s e r i e s c o n s is te d o f a s e r i e s w hich was s e t u p a s th e above b u t c o n ta in e d e s t r a d i o l L

15 r

-i b e n z o a te a s th e hormone t o be s tu d ie d * w hich c o n ta in e d

1*66

From a 1 cc* ampule

mg. i n sesam e o i l t h e r e was w ithdraw n

by means o f a s t e r i l e p i p e t t e 5 d ro p s whose volume was c a l ­ i b r a t e d so a s t o g iv e a c o n c e n tr a tio n o f *158% o f e s t r a d i o l b e n z o a te when added t o 2 .5 cc* o f p r o te o s e p eptone*

L ik e

am ounts o f sesam e o i l w ere added to 2 .5 cc* o f p r o te o s e p ep to n e*

L ik e am ounts o f sesam e o i l w ere added t o 2 .5 c c .

o f p r o te o s e p e p to n e i n t h r e e t e s t tu b e s and t h i s se rv e d a s th e c o n t r o l .

One o f th e 11 t e s t tu b e s i n th e e s t r a d i o l ben­

z o a te s e r i e s was p l a t e d on a g a r and in c u b a te d and i n l i k e m anner one o f t h e c o n t r o l s e r i e s was in c u b a te d .

The f o u r th

p a r t o f t h i s e x p e rim e n t c o n s is te d i n th e a d d i t i o n o f b o th t e s t o s t e r o n e and p r o g e s te r o n e t o 2 .5 c c . o f p r o te o s e pep­ to n e .

S t e r i l e p i p e t t e s w ere c a l i b r a t e d so a s t o o b ta in con­

c e n t r a t i o n s o f .175% p r o g e s te r o n e and .152$ t e s t o s t e r o n e when added to 2 .5 c c . o f p r o te o s e peptone*

L ik e am ounts o f

sesam e o i l w ere added t o 2 .5 c c . o f p r o te o s e p e p to n e i n 5 t e s t tu b e s .

As i n t h e above, one t e s t tu b e o f th e

11

i n th e

hormone s e r i e s and one o f t h r e e i n th e c o n tr o l s e r i e s was p l a t e d o n a g a r and in c u b a te d a t 37 d e g re e s G* t o check th e s t e r i l i t y o f th e e x p e rim e n ts . The above s e t o f e x p e rim e n ts was s e t up a s above and re p e a te d .

L

14

G en e ral rem arks on m ethods u s e d . The p h o to g ra p h y in v o lv e d i n t h i s s e t o f e x p e rim e n ts u s e o f h ig h sp eed f ilm and a v e ry s h o r t ex p o su re tim e,.

made T h is

m ethod was r e s o r t e d t o b ec a u se o f th e r a p id p la s m o p ty s is on th e p a r t o f th e o rg a n ism s upon rem oval from t h e i r medium*

Thus

i n o r d e r to p r e s e r v e t h e a c c u ra c y o f th e e x p e rim e n ts th e spec­ im ens w ere p h o to g ra p h ed w h ile s t i l l i n a v i a b l e a c t i v e s ta t e * The u s e o f m e t h y l - c e ll u l o s e , g ly c e r in e and o th e r n a r c o t i c s was fo u n d t o d i s t o r t th e o rg an ism s and hence was n o t u s e d a s a r o u t i n e p r o c e d u re . The gro w th c u rv e was d iv id e d i n t o th e fo llo w in g p e r io d s , ( H e n r ic i, O rd a l 1948)

5

t h e l a g p h a s e , d u rin g w hich th e c e l l s

m u ltip ly a t a n e v e r i n c r e a s i n g r a t e , u n t i l a p e r io d o f maxi­ mum r a t e o f grow th i s reach ed *

T h is i s th e lo g a r ith m ! c p h a s e .

T h is i s som etim es fo llo w e d by a p e r io d o f n e g a tiv e a c c e l e r a t i o n o f grow th r a t e , and i s th u s c a l l e d th e n e g a tiv e a c c e l e r a t i o n phase.

The r a t e o f gro w th a c c e l e r a t i o n may rem ain a t a max­

imum u n t i l th e gro w th peak i s re a c h e d and such a p e a k , i f th e p o p u la tio n i s c o n s ta n t f o r any a p p r e c ia b le l e n g t h o f tim e , i s te rm e d th e s t a t i o n a r y p h a s e .

A f te r t h i s , th e cu rv e tu r n s o v e r

i n t o th e p h a se o f a c c e l e r a t i n g d eath *

I n m ost c a s e s a f t e r

r e a c h in g a maximum d e a th r a t e , th e o rganism s c e a s e t o d ie so r a p i d l y , and one may re c o g n iz e a p h ase o f n e g a tiv e a c c e le r a ­ t i o n o f d e a th . L

15

III.

OBSERVATIONS AND RESULTS G e n e ra l

C olpidium campylum i s an e lo n g a te d re n ifo rm c i l i a t e . I t h a s been g e n e r a l l y d e s c r ib e d a s b e in g 50 t o 70 m ic ra i n le n g th *

The specim ens c u lt u r e d i n th e c o n tr o l s e r i e s i n

f a t - f r e e p r o te o s e p e p to n e w ere found to be 65 m ic ra i n le n g th and 50 m ic ra i n w id th ( f i g s . 1 , 2, and 5 ) .

T h is f ig u r e r e p ­

r e s e n t s th e mean m easurem ents o f 60 o rg a n ism s.

C o lp id ia i n

2 .5 cc* o f p r o te o s e p ep to n e showed a grow th c u rv e t h a t r e a c h ­ e s a h e i g h t o f 4 0 ,0 0 0 o rg an ism s a t th e 84 h o u r p e r io d a f t e r in n o c u la tio n ( f i g . 2 0 ) .

T here i s l i t t l e o r no l a g p h ase

p r e s e n t , how ever, when th e organism i s c u ltu r e d i n l a r g e r am ounts o f p r o te o s e p e p to n e medium ( S u lli v a n 1949) t h e r e i s a lo n g e r l a g p h a se p r e s e n t d ep en d in g upon th e amount o f medium used.

The lo g a r ith m ic p h a se e x te n d s up t o th e 72 h o u r p e r io d

a t w hich tim e t h e r e i s a mean p o p u la tio n o f 56 ,0 0 0 p e r c c . T here i s an e x tre m e ly s m a ll p e r io d o f n e g a tiv e a c c e l e r a t i o n w hich l a s t s o n ly tw e lv e h o u rs and i s fo llo w e d by a v e ry s h o r t p e r io d o f s t a t i o n a i y g ro w th d u rin g w hich tim e th e p o p u la tio n i s a t i t s maximum o f 4 0 ,0 0 0 p e r cc*

The p h a se o f a c c e le r a ­

t i n g d e a th i s s h o r t l i v e d a s w e ll and l a s t s o n ly

12

h o u rs

d u rin g w hich tim e th e p o p u la tio n s in k s to a l e v e l o f 28,000 per cc.

T h is i s fo llo w e d by a p e r io d o f n e g a tiv e d e a th

a c c e l e r a t i o n w hich i s q u i t e s h a rp a t i t s s t a r t and th e n l e v e l s o u t to m a in ta in a d ro p from 28,000 to 24,0 0 0 o rg an ism s p e r cc* d u rin g a 48 h o u r p e r io d th u s h o ld in g th e p o p u la tio n co u n t a t a 24,000 p e r c c . l e v e l f o r a n o th e r 24 h o u r s .

The p h a se o f

p o s i t i v e d e a th a c c e l e r a t i o n once a g a in en su es and a c o n s id e r ­ a b l e d ro p i n th e p o p u la tio n t a k e p la c e from th e s e v e n th day* s f i g u r e o f 24,000 p e r c c . to th e e le v e n th day* s f i g u r e o f 4 ,0 0 0 p e r cc*

T h ere i s a n o th e r l e v e l i n g o f f p e r io d o f n e g a tiv e d e a th

a c c e l e r a t i o n d u rin g w hich th e p o p u la tio n o f 4 ,0 0 0 p e r c c . i s m a in ta in e d f o r 24 h o u rs from th e e le v e n th day t o th e t w e lf th day.

A f te r t h i s tim e t h e r e i s a n o th e r p e r io d o f p o s i t i v e

d e a th a c c e l e r a t i o n w hich l a s t s f o r 48 h o u rs d u rin g w hich tim e t h e grow th l e v e l f a l l s from th e 4 ,0 0 0 p e r c c . l e v e l to 1 ,0 0 0 p er cc. S u c ro se s e r i e s Growth i n th e s u c ro s e medium a t th e end o f t e n t r a n s f e r s i n .1 M s u c ro s e medium r e a c h e s a h e ig h t o f 1 6 ,0 0 0 p e r c c . 5 d ay s a f t e r i n n o c u l a t i o n i n th e s u c ro s e medium.

The l a g p h ase

i s c o n s id e ra b ly e x te n d e d and l a s t s a b o u t 3 d a y s , a f t e r which th e lo g a r ith m ic p h a se e n su es and t h i s p h ase l a s t s

2

days a t

th e end o f w hich tim e a maximum o f 16,000 p e r c c . i s re a c h e d . T h e re i s a p p a r e n tly no p e r io d o f n e g a tiv e a c c e le r a tio n *

T here

i s lik e w is e no s t a t i o n a r y p h a se o f maximum grow th m a in ten an ce f o r a s soon a s th e maximum i s re a c h e d th e p e r io d o f a c c e l e r a t i n g L

_J

17

d e a th q u ic k ly e n su es and t h e r e i s a d ra m a tic drop from th e maximum l e v e l o f 1 6 ,0 0 0 t o 3 ,5 0 0 p e r c c . i n a m a tte r o f £4 h o u rs ( f ig * 19)*

T h is seems t o be r a t h e r g e n e r a l i n a l l grow th

s t u d i e s on C olpidium campylum ( E l l i o t t 1955)*

T h is sudden d ro p

i s fo llo w e d by a p e r io d o f n e g a tiv e a c c e l e r a t i o n i n d e a th r a t e and t h e r e i s a r i s e i n th e p o p u la tio n o f 4 ,0 0 0 o rg an ism s p e r c c . i n £4 h o urs*

T h ere i s a n o th e r r a p id f a l l a f t e r t h i s p e r­

io d o f some £ ,0 0 0 o rg an ism s p e r c c .

T h is i s fo llo w e d by a -

n o th e r a p p a re n t p e r io d o f n e g a tiv e a c c e l e r a t i o n so t h a t th e r e i s a n i n c r e a s e o f 1 ,0 0 0 o rg an ism s i n £4 h o u rs b u t t h i s i s f o l ­ low ed by a f a l l i n th e p o p u la tio n co u n t o f 1 ,0 0 0 o rg an ism s i n £4 hours*

The l e v e l o f 4 ,0 0 0 o rg an ism s p e r c c . i s h e ld

f o r £4 h o u rs and th e n a d ro p o f 500 o rg an ism s o c c u rs i n th e fo llo w in g £4 h o u r s .

The l e v e l o f 3 ,5 0 0 o rg an ism s i s h e ld f o r

£4 h o u rs and th e n a t th e 14 day p e r io d th e p o p u la tio n count d ro p s to th e l e v e l o f 1 ,0 0 0 p e r c c . w hich i s h e ld f o r 7£ h o u rs o r u n t i l th e end o f th e ex p erim en t# (Seaman and O 'M a lle y , 1 9 4 9 ). The p r o c e s s o f m o rp h o lo g ic a l a d a p ta tio n i n th e s u c ro s e medium was c a r e f u l l y stu d ie d *

The s tu d y in v o lv e d th e m easure­

m ent o f 30 o rg an ism s a t th e h e ig h th o f th e grow th p r o c e s s i n te n t r a n s f e r s from p r o te o s e p e p to n e to s u c ro s e a s o u tl i n e d i n t h e s e c t io n o f M a te r ia ls and M ethods*

T here was a r a t h e r gen­

e r a l d e c re a s e i n th e s i z e o f th e o rg an ism s u n t i l a mean f ig u r e o f 5£ m ic ra i n l e n g t h and 16 m ic ra i n w id th was re a c h e d ( f i g . £ 0 ) . T h is r e p r e s e n te d a d e c re a s e o f a lm o st J o f th e le n g th o f th e L

18 r

-i

specim en and e x a c tly § o f th e w idth*

The l o s s o f le n g th and

w id th was m ost pron o u n ced i n th e f i r s t h a l f o f th e t r a n s f e r s e r i e s , becoming more l e v e l e d o f f a s th e p r o c e s s went on ( f ig * 20)*

R o u tin e s t a i n i n g a t th e end o f th e a d a p ta tio n p ro ­

c e s s ( f ig * 7) d is c lo s e d a c l e a r l y s ta i n i n g m acro n u cleu s and m ic ro n u c le u s , w hich s to o d o u t i n c o n t r a s t to specim ens which had n o t been s ta i n e d ( f i g s . 4 , 6 ) .

I n th e s u c ro s e s e r i e s th e

f a t s u b s ta n c e a p p e a re d to be c o l l e c t e d aro u n d th e p e r ip h e ry o f th e specim en ( f i g . 7 ) , w h ile i n th e c o n tr o l s e r i e s , th e r e was a homogeneous d i s t r i b u t i o n o f f a t th ro u g h o u t th e e n t i r e organism ( f i g . 2 ) . The o rg an ism s w ere n o t a l t e r e d a s r e g a rd s t h e i r r a t e o f lo co m o tio n o r a b i l i t y to p erfo rm th e ’’a v o id in g ” r e a c t i o n c h a r a c t e r i s t i c o f th e p r o to z o a .

I n o r g a n ic s e r i e s Growth i n th e in o r g a n ic medium a t th e end o f te n t r a n s ­ f e r s re a c h e d a peak o f 1 8 ,0 0 0 org an ism s p e r c c . 6 days a f t e r in n o c u la tio n *

T h e re i s a r a t h e r d raw n -o u t l a g p h a se w hich l a s t s

48 h o u rs a t w hich tim e t h e mean p o p u la tio n i s 6 ,0 0 0 p e r c c . T h is i s fo llo w e d by a lo g a r ith m ic p h ase d u rin g which th e pop­ u l a t i o n r i s e s t o th e maximum o f 18,0 0 0 p e r c c .

T h ere i s no

p e r io d o f s t a t i o n a r y maximum grow th f o r a s soon a s th e grow th peak i s re a c h e d , t h e r e i s a sudden d ro p t o 6 ,0 0 0 o rg an ism s p e r

L

19

cc* i n t h e p e r io d o f p o s i t i v e d e a th a c c e l e r a t i o n ( f ig * 19)* T h is sudden d ro p i s fo llo w e d by a p e r io d o f n e g a tiv e a c c e l e r ­ a t i o n o f t h e d e a th r a t e and t h e r e i s a r i s e o f 2 ,5 0 0 o rg an ­ ism s p e r cc* i n 24 hours*

T h ere i s a n o th e r f a l l i n th e n e x t

24 h o u rs o f 2 ,5 0 0 organism s*.

T h is i s fo llo w e d by a n o th e r p e r­

i o d o f s l i g h t n e g a tiv e d e a th a c c e l e r a t i o n d u rin g w hich t h e r e i s a r i s e o f 1 ,0 0 0 organism s*

T here i s a g e n e r a l p o s i t i v e

d e a th a c c e l e r a t i o n f o r t h e n e x t 72 h o u rs t o th e l e v e l o f 5 ,0 0 0 o rg an ism s p e r cc* w hich i s h e ld f o r 24 h o u rs and t h e r e i s one l a s t r i s e 500 o rg an ism s b e f o re th e f i n a l drop to 5 ,0 0 0 o rg an ism s w hich co n clu d ed th e e x p e rim e n ts. The m o rp h o lo g ic a l changes o c c u r r in g d u rin g th e a d a p ta tio n t o th e in o r g a n ic medium from p r o te o u s p ep to n e w ere c a r e f u l l y s tu d ie d *

The s tu d y in v o lv e d th e m easurem ent o f 50 o rg an ism s

a t th e h e i g h t o f t h e grow th p r o c e s s i n 10 t r a n s f e r s from th e p e p to n e medium t o in o r g a n ic medium a s o u tl i n e d i n s e c t io n o f M a te r ia ls and Methods*

T h ere was a g e n e r a l d e c r e a s e i n th e

s i z e o f th e o rg a n ism s u n t i l a mean f ig u r e o f 48 m ic ra i n le n g t h and 52 mi e r a i n w id th was re a c h e d ( f ig * 20)*

T h is r e ­

p r e s e n te d a d e c r e a s e o f a lm o st one q u a r t e r th e l e n g th and w id th o f t h e o rg a n ism .

T h ere w ere p r e s e n t a ls o a g r e a t d e a l

o f rounded c y s t - l i k e form s w hich sp in n e d a b o u t i n a r a t h e r r e s t r i c t e d c i r c l e q u i t e v i o l e n t l y ( f ig * 5 ) .

The cy to p lasm o f

o rg an ism s i n t h e in o r g a n ic medium assum es a “r e f r a c t i v e ” ap­ p earan ce* L

The e n t i r e cy to p lasm a p p e a rs t o be f u l l o f many _l

20 ~i

d i f f e r e n t ty p e s o f c r y s t a l s w hich, when th e y come i n t o a p a th o f l i g h t * seem t o i l l u m i n a t e t h e i r cy to p lasm due to th e r e ­ f r a c t i o n phenomenon.

R o u tin e s ta in in g w ith L u g o lf s io d in e

s t a i n d i s c l o s e d th e p r e s e n c e o f a g r e a t number o f s ta r c h g r a n u le s and c r y s t a l s .

However, no c a r e f u l s t a t i s t i c a l s tu d y

was made t o s u b s t a n t i a t e any c la im t h a t i n th e in o r g a n ic me­ dium t h e r e i s an i n c r e a s e i n th e number o f c r y s t a l s p r e s e n t i n th e cy to p lasm o f C olm dium camnylum.

V itam in s e r i e s P r e lim in a r y o b s e r v a tio n s w ith th e B g ro u p o f v ita m in s p ro d u ced n e g a tiv e r e s u l t s i n grow th in c r e a s e s f o r n i a c i n , p y rid o x in e and c h o lin e c h l o r id e .

Those c o n c e n tr a tio n s o f

th e above t h r e e t h a t p ro d u ced th e h ig h e s t grow th l e v e l s even th ough th e y w ere u n d e r th e c o n tr o l l e v e l o f 40,000 f o r th e p e p to n e s e r i e s a r e shown i n f i g . 17* P r e lim in a r y o b s e r v a tio n s w ith a s c o r b ic a c id and FeClg gave n e g a tiv e r e s u l t s a s w ell*

Those c o n c e n tr a tio n s o f

a s c o r b ic a c i d and F eC l 3 t h a t p ro d u ced th e h i g h e s t grow th l e v e l s ev en th o u g h th e y d id n o t re a c h th e c o n t r o l l e v e l o f 4 0 ,0 0 0 f o r th e p ep to n e s e r i e s a r e shown i n f i g . 18* A s tu d y o f th e g ro w th cu rv e o f th e n i a e i n s e r i e s d i s ­ c lo s e s t h a t t h e maximum amount o f grow th a t t a i n e d i n th e optimum c o n c e n tr a tio n was u n d e r th e c o n tr o l f ig u r e o f 40 ,0 0 0

L

_l

21

by 4 ,0 0 0 organism s*. w ith n ia c in *

Thus n e g a tiv e r e s u l t s w ere o b ta in e d

I n th e *1% p y r id o x in e s e r i e s th e h e ig h t o f

g ro w th a t t a i n e d was 6 ,0 00 l e s s th a n th e c o n tr o l, th e re b y i l l u s t r a t i n g n e g a tiv e r e s u l t s *

The grow th h e ig h t i n th e

c h o lin e c h lo r id e s e r i e s optimum c o n c e n tr a tio n o f *1$ was 7 .0 0 0 l e s s th a n t h e c o n t r o l , th u s p ro v id in g n e g a tiv e e v id e n c e o f g ro w th s ti m u la tio n ( f ig * 17)* A s tu d y o f th e grow th cu rv e o f th e FeClg s e r i e s i n t h e optimum c o n c e n tr a tio n o f 1$ d is c lo s e s t h a t th e grow th h e i g h t i n th e optimum c o n c e n tr a tio n o f .0 1 $ a s c o r b ic a c id i s 1 5 ,0 0 0 l e s s th a n th e c o n tr o l ( f ig * 1 8 ) .

Optimum c o n c e n tr a tio n s tu d i e s The optimum c o n c e n tr a tio n o f th ia m in h y d ro c h lo rid e i n 2 .5 c c . o f p r o te o s e - p e p to n e was found to be .287$ (fig s * 15, 2 2 ).

The optimum c o n c e n tr a tio n was ta k e n as

t h a t c o n c e n tr a tio n t h a t p ro d u ced th e maximum amount o f grow th*

The sub-optim um c o n c e n tr a tio n o r t h a t c o n c e n tr a ­

t i o n w hich was l e s s th a n th e optimum amount o f c o n c e n tr a tio n and p ro d u ced t h e maximum amount o f grow th, was found to be .028$*

The supra-optim um c o n c e n tr a tio n o r t h a t c o n c e n tra ­

t i o n w hich was g r e a t e r th a n th e optimum and p ro d u ced th e maximum amount o f g ro w th , t h i s was found to be 1.42$* The grow th h e i g h t i n th e optimum c o n c e n tr a tio n was 1 2 0 .0 0 0 o rg an ism s p e r c c . o f medium, and was a t t a i n e d a t t h e e ig h th day*

The p o p u la tio n peak i n th e sub-optim um

zz

c o n c e n tr a tio n r e a c h e s 80,000 o rganism s p e r c c . o f medium and t h i s i s re a c h e d on th e f i f t h day*

The supra-optim um

c o n c e n tr a tio n re a c h e d i t s grow th h e ig h t o f 100,000 on th e e ig h th day ( f ig *

Z Z )*

The g e n e r a l c h a r a c t e r o f th e grow th p r o c e s s i n th e s e r i e s i s t h a t o f a bim odal curve* n o te ( f i g s * 1 5 ,

ZZ)

I t i s in te r e s tin g to

t h a t a s th e c o n c e n tr a tio n s d e c r e a s e

t h e r e i s a g r e a t e r and more ex ten d ed second peak*

Thus i n

t h e sub-optim um c o n c e n tr a tio n th e second peak l a s t s f o r

1Z

h o u r s , w h ile i n th e su pra-optim um c o n c e n tr a tio n th e second peak l a s t s b a r e ly 48 h o urs*

The lo w e rin g o f th e c o n c e n tra ­

t i o n seems t o e f f e c t th e tim e o f th e maximum grow th p e a k , th u s th e maximum peak o f th e optimum and supra-optim um con­ c e n tra tio n

i s re a c h e d on th e same day w h ile th e maximum o f

th e sub-optim um o c c u rs 72 h o u rs b e f o re o r on th e f i f t h day* The e n t i r e grow th p r o c e s s i s ex ten d ed o v e r a much g r e a t e r sp a ce o f tim e and th e f i n a l grow th l e v e l a t th e end o f th e e x p e rim e n t i s c o n s id e ra b ly r a i s e d p a r t i c u l a r l y a s r e g a rd s th e optimum c o n c e n tr a tio n w hich a t th e end o f 17 days main­ t a i n a grow th l e v e l o f 22 ,0 0 0 a s a g a i n s t th e c o n tr o l o f a z e ro re a d in g * The supra-optim um c o n c e n tr a tio n d i s p la y s an i n t e r e s t ­ in g s tu d y b ec au se o f th e s t a t i o n a r y p h ase t h a t i s m a in ta in e d a t th e h e i g h t o f th e grow th p ro c e ss*

W hile, s t r i c t l y s p e a k in g ,

no c o n s ta n t amount i s m a in ta in e d y e t th e d ro p o f o n ly 2 ,0 0 0 L

_!

23

o rg a n ism s p e r c c . a f t e r t h e maximum i s re a c h e d i s somewhat s i g n i f i c a n t s in c e i t d e m o n stra te s t h a t

i n t h i s concen­

t r a t i o n a t l e a s t , a c t s a s an a n ta g o n is t a g a in s t w h atev er sub­ s ta n c e may be r e s p o n s i b le f o r th e i n h i b i t i o n o f th e grow th p ro c e ss *

The w hole s tu d y o f

d e m o n s tra te s t h i s f a c t m erely

by th e e x te n s io n o f t h e grow th p r o c e s s , b u t t h e supra-optim um c o n c e n tr a tio n does so i n a p a r t i c u l a r way.

The a b i l i t y o f

t h e o rg an ism s to d i s p l a y a second peak and i n th e c a s e o f th e optimum c o n c e n tr a tio n a s l i g h t t h i r d peak m ust be i n t e r p r e t e d a s some s o r t o f a re c o v e ry from th e u s u a l e f f e c t s o f th e grow th i n h i b i t i n g s u b s ta n c e .

R ib o f la v in s e r i e s The optimum c o n c e n tr a tio n o f r i b o f l a v i n (Bg) i n 2 .5 c c . o f p r o te o s e p e p to n e was found to be *142%.

T h is c o n c e n tra ­

t i o n p ro d u ced a maximum g ro w th l e v e l o f 150,000 o rg an ism s p e r c c . t e n days a f t e r i n o c u l a t i o n .

The sub-optim um con­

c e n t r a t i o n was fo u n d t o be .014% w hich p ro d u ced a maximum g ro w th l e v e l o f 1 0 4 ,0 0 0 o rg an ism s p e r c c . 6 days a f t e r i n ­ n o c u la tio n .

The su pra-optim um c o n c e n tr a tio n was found t o

be 1.142% .

T h is p ro d u c ed a maximum grow th l e v e l o f 1 1 2 ,0 0 0

o rg a n ism s p e r c c .

The g e n e r a l e f f e c t o f r i b o f l a v i n i s t o

e x te n d th e gro w th p r o c e s s and t o p ro d u ce a s e r i e s o f second­ a r y s m a ll p ea k s o f g ro w th ( f i g s . 25, 1 5 ) .

L

24

T here i s no l a g p h ase p r e s e n t i n any o f th e c o n c e n tra ­ tio n s * A s tu d y o f th e optimum c o n c e n tr a tio n cu rv e shows a d e f i n i t e p h ase o f n e g a tiv e a c c e l e r a t i o n l a s t i n g from th e f i f t h t o th e e ig h th d a y .

T h is i s m ost m arked a t th e 7 to 8 day p e r ­

io d when t h e r e i s a r i s e o f o n ly 1 , 000 o rg a n ism s.

T h is i s

fo llo w e d by a lo g a r ith m ic p h a se w hich te r m in a te s a t th e max­ imum l e v e l o f 1 5 0 ,0 0 0 o rg an ism s p e r c c . a r y p h a se p r e s e n t .

T h ere i s no s t a t i o n ­

A p e r io d o f a c c e l e r a t i n g d e a th en su es

w hich e n d s on t h e t h i r t e e n t h day e n d i s fo llo w e d by a 24 h o u r p e r io d o f n e g a tiv e a c c e l e r a t i o n i n r a t e o f d e a th , so t h a t an ap p ro x im a te l e v e l o f 1 0 0,000 o rg an ism s i s h e ld f o r 24 h o u r s .

T h is i s fo llo w e d by a n o th e r p e r io d o f a c c l e r a t i n g

d e a th and th e l e v e l d ro p s t o 46,0 0 0 o rganism s p e r c c . a t th e end o f th e e x p e rim e n t. The supra-optim um c o n c e n tr a tio n cu rv e r e a c h e s a maximum grow th l e v e l o f 1 1 2,000 o rg an ism s p e r c c . and a d i s t i n c t post-maximum p e a k .

The la g p h a se i s a b s e n t and th e lo g a r ­

ith m ic p h a se e x te n d s u n t i l th e f i f t h day when a v e ry d i s ­ t i n c t n e g a tiv e p h a se o f grow th a c c e le ra tio n o c c u rs t h i s p e r io d e x te n d s from t h e f i f t h day to th e s i x t h day and i s fo llo w e d by a p e r io d o f p o s i t i v e grow th a c c e le ra tio n w hich c u lm in a te s i n th e maximum grow th l e v e l a t th e 7 day p e r io d .

T here i s

some p o s i t i v e d e a th a c c e l e r a t i o n b u t i n 96 h o u rs a f t e r th e maximum peak was re a c h e d t h e r e was o n ly a d rop o f 22,000

L

25 r

~i

org an ism s

a t t h e 11 day p e r io d , t h i s i s fo llo w e d by a nega­

t i v e d e a th a c c e l e r a t i o n so t h a t a r i s e o f 4 ,0 0 0 org an ism s i n 24 h o u rs i s se e n and th u s a s m a ll second peak i s produced on th e curve* e ra tio n .

T h e re i s f i n a l l y a p e r io d o f p o s i t i v e d e a th a c c e l­ The lo g a r ith m ic p h ase te r m in a te s a t th e maximum

peak v a lu e o f 1 0 4 ,0 0 0 o rg an ism s p e r c c . on th e s i x t h day a f t e r in n o c u l a t i o n .

T h is i s fo llo w e d by a d i s t i n c t a c c e l e r ­

a t i o n d e a th p h a se f o r 48 h o u rs d u rin g w hich t h e r e i s a drop o f 20 ,0 0 0 o rg a n ism s.

T h is p e r io d i s i n t u r n fo llo w e d

by a p h ase o f n e g a tiv e d e a th a c c e l e r a t i o n so t h a t a r i s e o f 4 .0 0 0 o rg an ism s i s g o tte n i n 24 h o u rs which i s fo llo w e d by a n o th e r p e r io d o f n e g a tiv e d e a th a c c e l e r a t i o n so t h a t a second p o s t maximum peak i s p ro d u c e d .

T h e re i s a n o th e r p e r io d o f

p o s i t i v e d e a th a c c e l e r a t i o n d u rin g which th e l e v e l d ro p s to a n o th e r 2 0 ,0 0 0 o rg an ism s and a c o n s ta n t v a lu e o f 64,000 o r ­ ganism s i s m a in ta in e d f o r 24 h o u r s .

T here i s a n o th e r p e r ­

io d o f p o s i t i v e d e a th a c c e l e r a t i o n and a n o th e r drop o f 20,000 o rg an ism s p e r c c . w ith a f i n a l p e r io d o f n e g a tiv e d e a th a c c l e r a t i o n r e s u l t i n g i n a t h i r d post-maximum peak and l a s t l y a p e r io d o f p o s i t i v e d e a th a c c e l e r a t i o n w ith th e l e v e l o f 4 0 .0 0 0 p e r c c . a s t h e f i n a l r e a d in g .

P a n to th e n ic a c id s e r i e s The optimum c o n c e n tr a tio n o f p a n to th e n ic a c id i n 2 .5 c c . o f p r o te o s e - p e p to n e was found to be .288%. L

T h is c o n c e n tr a tio n -I

26 r

n

p ro d u ced a maximum o f 1 0 2 ,0 0 0 o rg an ism s 14 days a f t e r in n o c u la tio n *

The supra-optim um c o n c e n tr a tio n was found to be

.0 2 8 $ w hich p ro d u ced a h e ig h t o f 76,000 o rg an ism s p e r cc* 16 d ays a f t e r in n o c u la tio n *

The supra-optim um c o n c e n tr a tio n

p ro d u ced a maximum o f 88,000 o rg an ism s 8 days a f t e r in n o c u ­ l a t i o n and was found t o be 1*288$ The g e n e r a l c h a r a c t e r o f th e grow th p ro c e s s i n t h i s s e r i e s i s one o f an e x te n s io n o f th e e n t i r e c y c le w ith th e r e s u l t t h a t a s e r i e s o f pre-maximum grow th peaks a r e g o t­ t e n b e f o re th e maximum h e ig h t i s a t t a i n e d .

The m a in ta in ­

in g o f a s t a t i o n a r y p h a se and a g e n e r a l l e v e l i n g o f th e g row th p eak s i s a n o th e r c h a r a c t e r i s t i c o f grow th i n t h i s s e r ie s * A s tu d y o f t h e optimum c o n c e n tr a tio n cu rv e d i s c l o s e s t h e a b se n c e o f any l a g p h a se ( f ig s * 15 and 24)*

The lo g a ­

r ith m ic p h ase t e r m in a te s a t th e 84 h o u r p e r io d w ith th e f i r s t peak o f grow th a t 4 2 ,0 0 0 p e r cc*

T h is i s fo llo w e d

by a p o s i t i v e d e a th a c c e l e r a t i o n p h ase w hich l e v e l s o f f from t h e f i f t h day to t h e s i x t h day so t h a t a l e v e l o f 2 8 ,0 0 0 p e r c c . i s m a in ta in e d f o r 24 h o u r s , t h i s p e r io d o f n e g a tiv e d e a th a c c e l e r a t i o n i s fo llo w e d by a p e r io d o f p o s i t i v e grow th a c c e l e r a t i o n w hich becomes maximum and h en ce may be term ed t h e lo g a r ith m ic p h a se o f grow th w hich p h a se p e r s i s t s u n t i l t h e se v e n th day o r f o r 72 hours*

A

p e r io d o f n e g a tiv e a c c e l e r a t i o n o f th e grow th p r o c e s s th e n L

27 r

ta k e s p la c e w hich p e r s i s t s u n t i l th e maximum o f 102,000 o rg an ­ ism s p e r c c . i s reach ed *

T h ere i s a r a p i d f a l l a f t e r th e max­

imum i s re a c h e d w ith th e o n s e t o f p o s i t i v e d e a th a c c e l e r a t i o n and no p e r io d o f n e g a tiv e d e a th a c c e le r a tio n * th e aforem en­ tio n e d f a l l te r m in a te s th e e x p e rim e n ts p ro d u c in g a l e v e l o f 32,000 o rg a n ism s p e r cc*

The p e r io d o f n e g a tiv e grow th ac­

c e l e r a t i o n b e f o r e th e maximum p ro d u ces a cu rv e t h a t i s t y p i ­ c a l o f b a c t e r i a and p ro to z o a i n g e n e r a l b u t uncommon i n th e C o lp id ia , i n t h a t a l l s t u d i e s on t h i s g e n e ra d is p la y a s h a rp r i s e and f a l l i n t h e i r grow th p a tte r n * T h e re i s no la g - p h a s e p r e s e n t i n th e supra-optim um c o n c e n tr a tio n gro w th c u rv e ( f ig * 24)*

T h ere i s a s h o r t l i v e d

p e r io d o f n e g a tiv e g ro w th a c c e l e r a t i o n fo llo w in g th e lo g a ­ r ith m ic p h a s e , and l a s t i n g f o r o n ly 24 h o u rs*

T h is p e r io d o f

n e g a tiv e grow th a c c e l e r a t i o n p re c e d e s th e pre-maximum peak o f 7 6 ,000 o rg an ism s w hich o c c u rs on t h e f o u r t h day a f t e r i n ­ n o c u la tio n *

F o llo w in g t h i s pre-maximum peak th e r e i s a

s h a rp d e c l i n e i n th e p o p u la tio n due to th e o n s e t o f th e p o s i t i v e d e a th a c c e l e r a t i o n , t h i s however l a s t s o n ly 24 h o u rs and i s i n t u r n fo llo w e d by a n e g a tiv e d e a th a c c e l e r a t i o n and re su m p tio n o f t h e lo g a r ith m ic p h a se u n t i l th e maximum i s re a c h e d on t h e e i g h t h day a f t e r in n o c u la tio n *

T here i s th e

u s u a l o n s e t o f p o s i t i v e d e a th a c c e l e r a t i o n w hich l a s t s f o r a p e r io d o f 5 d ay s d u r in g which t h e r e i s a d ro p o f 24,000 o rg a n ism s p e r cc* L

A p e r io d o f n e g a tiv e d e a th a c c e l e r a t i o n

_l

28 r

n ta k e s p la c e f o r 48 h o u rs so t h a t th e r e i s a r i s e o f some 4 .0 0 0 o rg an ism s from t h e t h i r t e e n t h t o th e f o u r te e n th day,. T h ere i s a f i n a l p e r io d o f p o s i t i v e d e a th a c c e l e r a t i o n and t h e e x p e rim e n t te r m in a te s on a l e v e l o f 12,0 0 0 p e r cc* T h ere i s no l a g p h a se p r e s e n t i n th e sub-optim um c o n c e n tr a tio n grow th curve*

The lo g a r ith m ic p h ase e x te n d s

from th e f i r s t day t o th e pre-maximum peak o f 4 4 ,0 0 0 p e r cc*

T h e re i s a 24 h o u r p e r io d o f p o s i t i v e d e a th a c c e le r a ­

t i o n w hich i s i n t u r n fo llo w e d by a 24 h o u r p e r io d o f n eg a­ t i v e d e a th a c c e l e r a t i o n w hich d u rin g th e l e v e l o f 4 0 ,0 0 0 o rg an ism s i s m ain ta in e d *

T h ere i s a n o th e r p e r io d o f p o s i­

t i v e d e a th a c c e l e r a t i o n w hich l a s t s f o r 48 h o u rs and t h i s i s fo llo w e d by an e x te n d e d lo g a r ith m ic p h a se w hich r e s u l t s i n t h e p r o d u c tio n o f t h e maximum peak a t th e 14 day p e r io d o f 7 6 .0 0 0 o rg an ism s p e r cc*

T h is maximum i s h e ld f o r 24 h o u rs

and th e n an e x te n d e d p o s i t i v e d e a th a c c e l e r a t i o n p h ase en­ s u e s and rem ain s u n t i l th e end o f th e ex p erim en t w hich term ­ i n a t e s on a l e v e l o f 2 0,0 0 0 o rg an ism s p e r cc*

Hormone s e r i e s An e x a m in a tio n o f t h e grow th cu rv e o f th e hormone s e r i e s , d i s c l o s e s th e f a c t t h a t th e g r e a t e s t amount o f grow th was a t t a i n e d i n th e p r o g e s te r o u s s e t o f ex p erim en ts*

The

grow th maximum i n t h i s s e t re a c h e d a h e ig h t o f 15 8 ,0 0 0 o r ­ ganism s p e r cc* on th e e ig h th day a f t e r in n o c u l a tio n i n L

_I

29 r

~ i

th e p r o g e s te r o n e s e r i e s *

The n e x t h i g h e s t grow th l e v e l

was a t t a i n e d i n th e t e s t o s t e r o n e s e r i e s , w hich re a c h e d a l e v e l o f 10 0 ,0 0 0 o rg an ism s p e r cc* on t h e f o u r te e n t h day a f t e r i n ­ n o c u la tio n *

The e s t r a d i o l s e r i e s was n e x t i n maximum a -

mount w ith 80,000 o rg a n ism s p e r c c . on th e t e n t h day a f t e r in n o c u la tio n .

The p r o g e s t e r o n e - te s to s te r o n e s e r i e s f o l ­

low ed n e x t w ith a g ro w th l e v e l o f 70,000 org an ism s on t h e e ig h th day a f t e r i n n o c u l a t i o n .

The c o n tr o l o f th e sesam e

o i l rem ain ed c o n s ta n tly th e same a s th e c o n tr o ls i n th e o th e r s e r i e s a p p a r e n tly d e n o tin g th e i n a b i l i t y o f th e o r ­ ganism t o u t i l i z e th e o i l to any e x te n t ( f i g . 16)* T h e re was no l a g p h a s e p r e s e n t i n any o f t h e s e t s o f e x p e rim e n ts ( f i g . 1 6 ) .

A ll o f th e s e t s fo llo w e d q u i t e

c l o s e ly th e p a t t e r n o f th e c o n tr o l up t o th e f o u r th day, a c h ie v in g a h e ig h t o f a b o u t 40,000 o rg an ism s a s a r e s u l t o f an e x te n d e d lo g a r ith m ic p h a s e .

A f te r th e f i r s t peak i s

re a c h e d t h e c h a r a c t e r o f th e cu rv es changes r a t h e r c o n s id e r ­ a b ly .

The p r o g e s te r o n e cu rv e u n d erg o es a p e r io d o f p o s i t i v e

d e a th a c c e l e r a t i o n w hich i s s h o r t l i v e d and a f t e r 48 h o u rs a n e x te n d e d lo g a r ith m ic p h a se en su es w hich te r m in a te s a t t h e maximum peak o f 1 5 8 ,0 0 0 org an ism s p e r c c .

T h is peak i s

fo llo w e d by a s h a rp d ro p i n th e p o p u la tio n cu rv e i n 48 h o u rs w hich i s th e r e s u l t o f a v e ry p o w e rfu l p o s i t i v e d e a th a c ­ c e le r a tio n phase.

F o r a f i v e day p e r io d a f t e r t h i s f a l l o f

some 7 0 ,0 0 0 o rg an ism s a c o n s ta n t l e v e l o f 70,000 o rg an ism s L

50

i s m a in ta in e d .

T h e re i s a f i n a l p h ase o f p o s i t i v e d e a th a c ­

c e l e r a t i o n w hich te r m in a te s th e e x p erim en ts on a l e v e l o f 50,000 o rg a n ism s. The grow th cu rv e i n th e t e s t o s t e r o n e s e r i e s i l l u s t r a t e s th e a b sen c e o f a l a g p h a se and th e summation o f a lo g a ­ r ith m ic p h a se w ith th e p r o d u c tio n o f a pre-maximum peak o f 5 7 ,0 0 0 o rg an ism s p e r c c .

T h is i s fo llo w e d by a b r i e f

p e r io d o f p o s i t i v e d e a th a c c e l e r a t i o n and th e n a p e r io d o f l e v e l i n g o f f due t o th e o n s e t o f a n e g a tiv e d e a th a c c e l e r a ­ tio n phase.

A n o th er s h o r t p e r io d o f p o s i t i v e d e a th a c c e le r a ­

t i o n ta k e s p la c e and a g a in i s fo llo w e d hy a l e v e l i n g o f f p e r io d so t h a t t h e p o p u la tio n

i s h e ld a t 22,000 f o r 24 h o u rs .

T h ere i s a r a p i d r i s e due t o th e o n s e t o f a n o th e r lo g a r ith m ic p h a se w ith th e p o p u la tio n r e a c h in g a f ig u r e o f 50,000 o rg an ­ is m s .

T h e re i s a f a l l o f 28,000 organism s i n 24 h o u rs due

t o an a c t i v e p o s i t i v e d e a th a c c e l e r a t i o n p h a s e .

T here i s a su b ­

s e q u e n t r i s e o f e x a c t l y th e same amount t h a t f e l l i n 24 h o u rs , i . e . , 2 8 ,000 o rg a n ism s, how ever t h i s re c o v e ry ta k e s 72 h o u r s , so t h a t a t t h e 12 day p e r io d t h e r e i s a g a in a p o p u la tio n r e a d in g o f 50 ,0 0 0 o rg a n ism s.

T here i s one f i n a l lo g a r ith m ic

p h a se t h a t r e s u l t s i n th e maximum h e ig h t o f 1 0 0 ,0 0 0 o rg an ­ is m s .

T h is i s fo llo w e d by a r a p i d f a l l and a l e v e l i n g o f f

and th e e x p e rim e n t ends w ith a l a s t f a l l o f th e grow th l e v e l t o 80,000 o rg an ism s p e r c c .

L

51

The grow th c u rv e i n th e e s t r a d i o l s e r i e s p r e s e n ts an o v e r - a l l smooth and l e v e l appearance*

T h is i s e s p e c i a l l y

e v id e n t i n th e f i r s t pre-maximum peak w hich i s a t t a i n e d on th e t h i r d day and a l e v e l o f 5 6 ,0 0 0 o rg an ism s p e r c c , i s m a in ta in e d f o r £4 h o u r s . o rg an ism s i n 48 h o u rs

T here i s a d ro p o f a lm o st £0,000

due to th e p o s i t i v e d e a th a c c e le r a ­

t i o n p h a se b u t t h e c u rv e r a p i d ly r i s e s to p ro d u ce a maximum peak o f 80,000 o rg an ism s i n 5 d a y s .

T h e re i s a su b se q u e n t

d ro p o f 25,000 o rg a n ism s i n 48 h o u rs due to th e p o s i t i v e d e a th a c c e l e r a t i o n r a t e i n c r e a s e , t h i s i n t u r n fo llo w e d by a s l i g h t r i s e due t o a r e d u c tio n i n th e r a t e o f p o s i t i v e d e a th a c c e le ra tio n .

T h e re i s a r a p id and m arked r i s e i n th e r a t e

o f p o s i t i v e d e a th a c c e l e r a t i o n so t h a t i n a m a tte r o f 48 h o u rs t h e r e i s a f a l l o f some 4 0 ,0 0 0 o rg a n ism s,

A la s t

s l i g h t peak i s a t t a i n e d due t o a r e d u c tio n i n th e r a t e o f p o s i t i v e d e a th a c c e l e r a t i o n w ith a r i s e o f 2 ,0 0 0 o rg an ism s i n £4 h o u rs and th e ex p erim en t ends w ith a f a l l o f £ ,0 0 0 o rg a n ism s so t h a t t h e l e v e l o f 57,0 0 0 o rganism s i s a t t a i n e d . The p r o g e s t e r o n e - t e s t o s t e r o n e cu rv e p r e s e n ts a r a t h e r sm ooth and l e v e l ap p earan ce*

I t a t t a i n s a pre-maximum

h e i g h t o f 4 6 ,0 0 0 o rg a n ism s p e r c c . and t h i s i s fo llo w e d by a d ro p o f n e a r ly 1 0 ,0 0 0 o rg an ism s i n £4 hours*

From th e f o u r th

day t o t h e s e v e n th day th e r e i s a p e r io d o f lo g a r ith m ic g ro w th w ith th e a tta in m e n t o f 70,000 o rg an ism s p e r c c . a s th e

L

s2

grow th h e ig h ts

T h is i s fo llo w e d by a r a p i d o n s e t o f p o s i t i v e

d e a th a c c e l e r a t i o n so t h a t i n 24 h o u rs th e r e i s a f a l l o f some 2 4 ,0 0 0 organism s#

T h ere i s a l e v e l i n g o f f p e r io d and

th e n a f t e r m a in ta in in g th e co u n t o f 50,000 organism s f o r 24 h o u r s , a lo g a r ith m ic p h a s e en su es and a f t e r 96 h o u rs th e max­ imum peak o f 72,000 o rg an ism s i s re a c h e d .

T here i s a l a s t

f a l l o f 1 5 ,0 0 0 o rg an ism s and th e n a l e v e l i n g o f f p e r io d ta k e s p la c e f o r a n o th e r 24 h o u rs o r u n t i l th e end o f th e e x p e rim e n t, w hich ends on a l e v e l o f 6 8 ,0 0 0 o rg an ism s p e r c c , A s tu d y o f th e m o rp h o lo g ic a l changes i n th e hormone s e r i e s d i s c l o s e s th e f a c t t h a t t h e g r e a t e s t in c r e a s e i n s i z e t a k e s p la c e i n th e t e s t o s t e r o n e s e r i e s ( f ig # 21)#

I n th a t

s e r i e s a t th e se v en day p e r io d , t h e r e i s an in c r e a s e o f 4 m ic ra i n t h e mean m easurem ents o f th e le n g t h o f 60 o rg an ism s and a n i n c r e a s e o f 3 m ic ra i n t h e i r w id th .

A t th e 16 day

p e r io d o f grow th i n t e s t o s t e r o n e , th e r e i s an i n c r e a s e o f 25 m ic ra i n th e l e n g th o f th e org an ism s and an i n c r e a s e o f 1 m icro n from th e mean m easurem ents o f th e c o n t r o l.

The t r e ­

mendous i n c r e a s e i n s i z e o f th e o rg an ism s r e s u l t s i n th e p r o d u c tio n o f " g ia n t" org an ism s ( f i g s . 1 1 , 1 2 ) , which a r e f u l l o f v a r io u s c r y s t a l s , and a p p e a r norm al i n e v e ry r e s p e c t except s iz e . I n p ro g e s te r o n e and e s t r a d i o l t h e r e i s a g e n e r a l de­ c r e a s e i n b o th th e l e n g t h and w id th o f th e o rg an ism .

L

I n th e

_l

33

c a s e o f t h e p r o g e s te r o n e s e r i e s th e mean m easurem ents a t th e 7 day p e r io d a r e 50 m ic ra lo n g and 28 m ic ra w id e ; t h i s r e p r e ­ s e n ts a d e c r e a s e o f 15 m ic ra i n th e le n g th o f th e organism and a d e c r e a s e o f 4 m ic ra i n th e width*

The m easurem ents a t

th e 16 day p e r io d a r e 63*6 m ic ra lo n g and 28*8 m ic ra wide* The g e n e r a l e f f e c t a s r e g a r d s th e s iz e a p p e a rs to be th e p ro ­ d u c tio n o f t r i a n g u l a r shaped org an ism s whose c i l i a r y m e rid ia n s a p p e a r t o be in c r e a s e d i n num ber; no a c t u a l c o u n t however was made on any sound s t a t i s t i c a l b a s i s .

I n th e s e t r i a n g u l a r

shaped o rg an ism s ( f i g . 13) t h e a n t e r i o r end a p p e a rs p o in te d and e n la r g e s o u t t o form a b ro ad p o s t e r i o r end.

The o rg an ­

ism s a p p e a r q u i t e norm al i n r e s p e c t to c y to p la sm ic c o n s t i t u ­ e n ts and lo co m otion* I n th e e s t r a d i o l s e r i e s ( f ig * 10) a t th e 7 day p e r io d th e r e i s a d e c r e a s e o f 24 m ic ra i n th e le n g th o f th e organism and 11 m ic ra i n th e w idth*

A t th e 16 day p e r io d t h e r e i s a

d e c re a s e o f 14 m ic ra i n t h e l e n g th and 8 m ic ra i n th e w id th o f th e o rg an ism i n com parison w ith th e c o n tr o l*

Sane t r i ­

a n g u la r form s a r e p r e s e n t h e r e a s i n th e p r o g e s te r o n e s e r ie s *

L

54 r

E x p la n a tio n o f F ig u re s 1*

P h o to m icro g rap h showing C olpidium campylum i n th e c o n tr o l s e r i e s o f

p r o te o s e p e p to n e .

S u p er-im p o sed s c a le h a s a v a lu e o f 1 .5 m ic ra f o r each g r a d a tio n .

L e n g th o f a specim en 65

m ic ra ; w id th 50 m ic ra .

2.

M a g n if ic a tio n X 4 5 0 .

P h o to m icro g rap h o f C olpidium campylum i n th e medium o f c o n tr o l s e r i e s a f t e r b e in g s ta i n e d w ith .0 6% Sudan B lack , t o i l l u s t r a t e th e even d i s t r i b u t i o n o f f a t .

L_

M a g n if ic a tio n X 450.

-j

56 r

1

E x p la n a tio n o f F ig u re s

5*

P h o to m icro g rap h o f C olpidium campylum a f t e r a d a p ta tio n i n th e I n o r g a n ic medium.

The

specim en i n t h e c e n te r o f th e f i e l d m easu res 48 mi e r a i n le n g th and 25 m ic ra w id e .

The

s p h e r i c a l e n c y s te d form m easures 27 m ic ra i n d ia m e te r .

M a g n if ic a tio n X 4 5 0 .

Each g ra d a ­

t i o n on superim posed s c a l e h a s a v a lu e o f 1 .5 m ic ra .

4.

P h o to m icro g rap h o f C olpidium campylum a f t e r a d a p ta tio n i n th e S u c ro se medium.

The mean

m easurem ents i n t h i s s e r i e s w ere 32 m ic ra i n l e n g t h and 16 m ic ra i n w id th . tio n X 450.

M a g n ific a ­

Each g r a d a tio n on su p erim p o sed

s c a l e h a s a v a lu e o f 1*5 m ic ra .

-j

38 r

E x p la n a tio n o f F ig u re s 5*

P h o to m icro g rap h o f C olpidium campylum i n t h e c o n tr o l medium o f 3% f a t - f r e e p r o te o s e peptone*

The specim ens m easure 65 m ie ra

lo n g and 30 m ie ra w ide.

Each g r a d a tio n on

su perim posed s c a l e e q u a ls 8 .1 mi e r a .

Mag­

n i f i c a t i o n X 120.

6.

P h o to m icro g rap h o f C olpidium campylum a f t e r a d a p ta tio n i n th e S u cro se medium.

The mean

m easurem ents a r e 32 mi e r a i n l e n g th and 16 mi e r a i n w id th .

Each g r a d a tio n on su perim posed

s c a le e q u a ls 8 .1 m ic ra . X 120.

L

M a g n if ic a tio n

40 r

E x p la n a tio n o f F ig u re s 7.

P h o to m icro g rap h o f C olpidium campyium s ta i n e d w ith .06% Sudan Black*

The specim ens were

ta k e n from S u cro se s o l u t i o n and r e p r e s e n t t y p i c a l specim ens a f t e r grow th i n th e s u c ro s e medium f o r 50 days*

They m easure

52 m ic ra i n l e n g th and 16 m ic ra i n w idth* Each g r a d a tio n on superim posed s c a l e e q u a ls 8 .1 m ic ra .

8*

M a g n ific a tio n X 120.

P h o t ami c ro g rap h o f C olpidium campyium show­ in g th e m o rp h o lo g ic a l changes d u r in g th e f i r s t h a l f o f th e grow th s tu d ie s i n th e P ro g e s te r o n e s e r ie s *

The mean d ia m e n tio n s

i n t h i s s e r i e s a r e 50 m ic ra lo n g and 28 m ic ra wide*

Each g r a d a tio n on su p erim p o sed

s c a l e e q u a ls 8 .1 m icra*

M a g n if ic a tio n

X 120*

L_

-J

41 _i

42 r

i

E x p la n a tio n o f F ig u re s 9.

P h o to m icro g rap h o f C olpidium campyium showing t h e m o rp h o lo g ic a l changes d u rin g t h e f i r s t h a l f o f th e grow th s t u d i e s i n th e t e s t o s t e r o n e s e rie s *

The mean m easurem ents i n t h i s s e r i e s

w ere 69 m ic ra lo n g and 55 m ic ra wide* c a tio n X 120.

M ag n ifi­

Each g r a d a tio n oifc th e superim ­

p o sed s c a l e e q u a ls 8*1 m icra*

10.

P h o to m icro g rap h o f C olpidium campyium showing t h e m o rp h o lo g ic a l changes d u rin g t h e f i r s t h a l f o f t h e grow th s tu d i e s i n th e E s t r a d i o l s e r ie s *

The mean m easurem ents i n t h i s s e r i e s

w ere 41 m ic ra lo n g and 21 m ic ra wide* c a tio n X 100* 11*7 m icra*

L_

M ag n ifi­

Each g r a d a tio n on s c a l e e q u a ls

43 r

44

r

“i

E x p la n a tio n o f F ig u re s 11.

P h o to m ic ro g rap h o f C olpidium campyium show­ in g th e m o rp h o lo g ic a l changes d u rin g th e seco n d h a l f o f th e grow th s tu d i e s i n th e te s to s te ro n e s e r ie s .

The mean m easure­

m ents i n t h i s s e r i e s were 90 m ic ra lo n g and 33 m ic ra wide* 8 .1

12.

m ic ra .

M a g n ific a tio n X 120.

The same a s aboisre, showing more o f th e “g i a n t ” fo rm s.

!_

Each g r a d a tio n e q u a ls

45

( -

VC".-j

F ig . L.

12

46 r

E x p la n a tio n o f F ig u r e s 15,

P h o to m ic ro g rap h o f C olpidium campyium show ing t h e m o rp h o lo g ic a l changes d u rin g t h e seco n d h a l f o f th e grow th s tu d i e s i n th e p r o g e s te r o n e s e rie s *

The mean m easurem ents a r e 63*& n d c ra

lo n g and 2 8 .8 m ic ra wide* e q u a ls 8 .1 m icra*

14*

Each g r a d a t io n

M a g n if ic a tio n X 120*

P h o to m ic ro g ra p h o f ColpidiuBi campyium show­ in g t h e m o rp h o lo g ic a l changes d u rin g th e second h a l f o f t h e grow th s tu d i e s i n th e e s t r a d i o l s e rie s .

The mean m easurem ents a r e 5 1 .2 m ic ra

i n l e n g t h and 2 4 .5 m ic ra i n w id th . a t i o n e q u a ls 8 .1 m ic ra .

L.

Each g ra d ­

M a g n if ic a tio n X 12 0 .

-J

47 r

L .

Explanation o f Figure Graph showing th e g ro w th o f C olpidium campyium i n th e optimum c o n c e n tr a tio n s o f th e v ita m in se rie s* . th e c o n tro l s e r ie s i n p ep to n e*

Curve (_______) , Z%

f a t - f r e e p r o te o s e

Curve ( • • • » • • ) th e T hiam in

Hydro c h lo r id e s e r ie s * t h e R ib o f la v in se rie s* . t h e P a n to th e n ic s e r ie s *

Curve (__ • Curve (__________

.) )

49

160.000

140,000

120,000

LOO,000

80,000

2 0,0 0 0

Jl___ Xo. of Dayc After Innoculation L.

F ig , 15

14

16

17

50 r

1

E x p la n a tio n o f F ig u re 16.

Graph showing th e grow th o f C olpidium campyium i n th e hormone s e r i e s . c o n tr o l s e r i e s i n

5%

sesame o i l s o lu t i o n . te r o n e s e r i e s . s e rie s .

Curve (________) , th e

f a t - f r e e p r o te o s e p e p to n e Curve ( • • • • • • ) th e p ro g e s ­

Curve (

Curve (___ .

>, th e t e s t o s t e r o n e . ) , th e e s t r a d i o l s e r i e s .

Curve ( __________ ) th e p ro g e s te r o n e ^ -te s to s te r o n e s e rie s .

- i

51 r

1 4 0 ,0 0 0

1 2 0 , 00C

100,000

8 0 , OOC

v § 4 0 , OOC U)

&20,OOC

1

A

5

6

7

8

9

10

11

12

13

N o . o f D ays A f t e r I n n o c u l a t i o n * 16

L

14

15

16 1 7

Explanation o f Figure Graph show ing th e grow th o f C olpidium campyium i n v a r io u s m edia.

Curve (_________) th e con­

t r o l s e r i e s o f 2 .5 p r o te o s e p ep to n e w ith o u t th e added v ita m in s u b s ta n c e s . th e

1%

n ia c in s e r ie s .

Curve ( . . . . • » » * )

Curve (_______ ) th e

♦1% p y r id o x in e s e r i e s . c h o lin e c h l o r id e s e r i e s .

C u r v e _______ ) t h e

\%

55 I

r

4 0 ,000

No. of Organisms

in

30,000.

•- -

2 0 ,0 0 0

1 0 ,0 0 0

o

1

2

3

4

5

6

7

8

9

10

11

No. of Days After Innoculatlon

Fig* 17

12

13

14 15

16

54 r

E x p la n a tio n o f F ig u re 18*

Graph showing th e grow th o f C olpidium campyium

i n v a r io u s media*

Curve (

),

th e c o n t r o l s e r i e s o f 2*5 p r o te o s e peptone* Curve (__________ ) th e

1%

FeClg medium*

Curve ( _______ ) , th e .0 1 # a s c o r b ic med­ ium*

L.

-J

55

r

No. of Organisms in 1 cc.

40.000

30,000

2 0 ,0 00

1 0 ,0 0 0

13

01

No. o f Days A fte r I n n o c u la tio n

L.

“i

Explanation o f Figure Graph show ing th e grow th o f C olpidium campyium i n v a r io u s media* tr o l s e rie s o f (

Z%

Curve (______ ) , th e con­ p r o te o s e p e p to n e .

* __)* th e in o r g a n ic s e r ie s * .

( * .. * • * * ) , th e

*1

Curve

Curve

M. s u c ro s e s e r ie s *

57 ~i

r

No. of Organisms

in 1 cc.

40,000

30,000

20,000

10,000 * r ''V 0

0

1

2

3

4

5

6

7

8

9

10

11

12 13

No. o f Days A f te r I n n o c u la tio n \

F i* . 1 0

L _

14

15 16 17

Explanation o f Figure I l l u s t r a t e s th e m o rp h o lo g ic a l changes t h a t ta k e p la c e d u rin g th e a d a p ta tio n p r o c e s s . The ( ______ ) l i n e i n d i c a t e s th e c o n tr o l le n g t h and w id th i n m ic ra .

The (__ . __ )

l i n e i n d i c a t e s in o r g a n ic s e r i e s l e n g th and w id th .

The ( .............) l i n e i n d i c a t e s

th e .1 M. s u c ro s e le n g th and w id th .

Each

p o i n t r e p r e s e n ts th e mean m easurem ents o f 30 o rg an ism s i n th e c a se o f th e in o r g a n ic and s u c ro s e medium and o f 60 o rg an ism s i n th e c o n tr o l s e r i e s .

59

70

60

50

Measurements

in Micra.

40

30

~0 ^

20

10

10 N o. o f T ra n s fe rs

L _

Explanation o f Figure I l l u s t r a t e s th e m o rp h o lo g ic a l changes t h a t accompany th e grow th o f C olpidium campyium i n th e hormone s e r i e s .

The (

)

lin e

i n d i c a t e s th e le n g th and w id th o f th e c o n tr o l s e rie s .

The ( . . . . . . )

l i n e i n d i c a t e s th e

l e n g t h and w id th i n th e t e s t o s t e r o n e s e r i e s . The (__________ ) , l i n e i n d i c a t e s th e grow th i n th e e s t r a d i o l s e r i e s .

The (

•

♦)

l i n e i n d i c a t e s th e grow th i n th e p r o g e s te r o n e s e rie s .

Each p o i n t r e p r e s e n t s th e mean meas­

u rem en ts o f 30 o rg an ism s i n th e hormone s e r ­ i e s and 60 o rg an ism s i n th e c o n tr o l s e r i e s .

61

90

80

70

60

50 C3 f-i o 40 •H

30 CO Cl a s

20

10

TTo .

of

D ays A f t e r I n n o d i l a t i o n

Explanation o f Figure

Graph show ing th e grow th o f C olpidium campyium i n t h e th ia m in e h y d ro c h lo rid e s e r ie s * (______ ) t h e c o n tr o l s e r i e s o f p e p to n e .

Curve (

c e n t r a t i o n s e r ie s *

..

5%

Curve

p r o te o s e

) th e optimum con­

Curve ( _ ________) th e

s u p ra optimum c o n c e n tr a tio n s e r i e s .

Curve

(__________ ) th e sub optimum c o n c e n tr a tio n s e rie s .

65

1 4 0 ,0 0 0

L 2 0 ,0 0 0

LOO,000

3 0 ,0 0 0

/

A

6 0 ,0 0 0

40,000 •H

No- o f Days A fte r I n n o c u la tio n

Explanation o f Figure Graph showing th e grow th o f C olpidium campyium i n th e r i b o f l a v i n s e r ie s * t h e c o n tr o l s e r i e s o f Curve ( ___ •• s e r ie s *

5%

Curve ( _______) , p r o te o s e p eptone*

) th e optimum c o n c e n tr a tio n

Curve (_________ ) th e s u p ra optimum

c o n c e n tr a tio n s e r ie s *

Curve (__________ )

th e su b optimum c o n c e n tr a tio n s e r ie s *

65

140,000

120,000

100 ,0 0 0

80,000

60,000

40,000

No. o f Days A fte r In n o c u la tio n

Explanation o f Figure

Graph showing t h e grow th o f C oloiditua campylum i n th e p a n to th e n ic a c id s e r i e s . th e c o n tr o l s e r i e s o f Curve ( __ »• s e rie s .

Z%

C urve (_______) ,

p r o te o s e p e p to n e .

) , th e optimum c o n c e n tr a tio n

Curve (

) , th e s u p ra op­

timum c o n c e n tr a tio n s e r i e s .

Curve ___

t h e sub optimum c o n c e n tr a tio n s e r i e s .

)

67

140,000

120,000

100,000

80,000

60,000

H4 0 ,000 •H

•H

1

o

6

7

10 11 12 IS 14 Mo, o f D ay s A f t e r I n n o c u l a t i o n

8

9

15

16

17

Explanation o f Figure (1 ).

Scheme r e p r e s e n tin g th e p o s s i b l e m e ta b o lic

pathw ays o f th e grow th p r o c e s s i n th e S u c ro se s e r ie s * l ( ? ) * The breakdown o f s u c ro s e w hich b e g in s th e m e ta b o lic p r o c e s s , presum ably by th e u s e o f an enzyme sy stem , e . g . , s u c r o s e . t i o n o f g ly co g en

by

2 (G ), th e form a­

th e p r o c e s s o f g ly c o g e n e s is .

3 ( P ) , t h e fo rm a tio n o f p y ru v ic a c id by th e p ro ­ c e s s o f p h o s p h o ry la tio n .

4 (C ), th e fo rm a tio n o f

f a t from a c e t i c a c id by th e p r o c e s s o f condensa­ t i o n o f th e in te r m e d ia r y p ro d u c t o x a lo a c e tic a c i d . 5 (C ), th e second pathw ay f o r th e fo rm a tio n o f f a t , by th e c o n d e n s a tio n o f a c e t i c a c id and th e d i r e c t p ro d u c tio n o f f a t t y a c id s w ith o u t an in te r m e d ia r y p r o d u c t.

6

P . th e fo rm a tio n o f p r o t e in from th e

tra n s d e a m in a tio n o r d e a m in a tio n o f th e a lp h a k e to a c id s i n th e K reb c y c l e .

r e p r e s e n ts th e

s te p s i n th e t r i c a r b o x y l i c a c id c y c le . (2 ).

Scheme r e p r e s e n tin g th e p o s s ib le m e ta b o lic

pathw ays o f th e grow th p r o c e s s i n th e a c e t a t e med­ ium .

I , th e fo rm a tio n o f p r o t e i n and c a rb o h y d ra te

d i r e c t l y from a c e ta te * a c id s from a c e ta te *

I I , th e fo rm a tio n o f f a t t y

I I I , th e fo rm a tio n o f f a t t y

69

CARBOHYDRATE F u m a r ic A c id

G ly c o g e n P’R OTFIN — \ X e rin e \ G lu c o s e

G lu c o s e |f ^ G lu c o s e

1

p h o s p h a te

6

p h o s p h a te

l^ c in e | V a lin e I -JH u c o se C y s te in e

F r u ^ t^ s e d ip h o s p h a te

P y r u & i t e A c e r y p n r o s p h a te

e)

O x a lo a c e ta te

G ly c e ro l \ F a t t y A c id J

________

M a la ^ e

FAT

PROTFIN A c e to a c e ta te ;

C is -A ^ o n ita te

,e u c m e Is o le u c in e ‘T y r o s i n e tPh e n y 1a1ani ne

T .Ialic A c id 6 P Gl u t a m i c A lp h a -k e to g lu ta ric ! A c id f ------ A c id i 6 p As p a r t i c ^ ^ T' A c id ; O x a l o a c e t i c A c id O x a l o 's u c c i n i c . A c id 4 (C ) \ P A la n in e )

A l p)]4a i f a-]- k e t o g l u t a r a t e k - G l u t a n i i c A c id ou

c c in a te

/A rg in in e P ro lin e f H is tid in e O rn ith in e

P y r u v i c —> A c e t i c A c id * A c id

fs(p)

G ly c o g e n

^ A c o n itic A c id -

Is o -c itric > A c id

(C) F a t t y A c id s

2 (G )

Is o c itra te

P u m a r a te

S u c c i n i c A c id fr

G iu c o s e (1 )

BGCROGF ACETATE

C a rb o h y d ra te VI (3 )

II P ro te in

IV

V II

III Fatty A c id

F ig .

V.5

70 r

a c id s from p r o t e i n .

IV , th e fo rm a tio n o f

c a rb o h y d ra te from f a t t y a c i d s .

V, th e form a­

t i o n o f c a rb o h y d ra te from p r o t e i n .

V II, th e

fo rm a tio n o f f a t t y a c id s from c a rb o h y d ra te . (5 )

Scheme r e p r e s e n tin g th e i n t e g r a t i o n

o f p r o t e i n , f a t and c a rb o h y d ra te m etab o lism , w ith th e p r i n c i p l e s u b s ta n c e s in v o lv e d a s o u t­ l i n e d i n ( l ) and

L

(2 )

o f th is f ig u re .

Explanation o f Figure Scheme r e p r e s e n tin g th e r o l e p la y e d i n t h e m etab o lism o f t h e C olpjdium campylum by th e v a r io u s v ita m in s t h a t p ro d u ced i n c r e a s e s i n th e gro w th

o f

th e organism s*

(R ), r e p r e s e n ts th e s u b s ta n c e R ib o f la v in and i n t h e f ig u r e i t i s a lo n g s id e o f th e s u b s ta n c e whose p r o d u c tio n i t i s co n cern ed w ith a s t h e f l a v i n enzyme. ( T ) , r e p r e s e n ts th e s u b s ta n c e Thiam ine and i t s c o n n e c tio n w ith th e c a rb o x y lm tio n d e c a r b o x y la tio n o f p y ru v ic a c id . ( P ) , r e p r e s e n ts th e s u b s ta n c e P a n to th e n ic a c id and i l l u s t r a t e s th e u s e o f th e a la n in e p o r tio n o f th e P a n to th e n ic a c id m o lecu le i n t h e m e ta b o lic p r o c e s s .

2

PROTEIN

CAREOHYDRATE

Serine (R) I Glycine (R)

fa:

^—G I y cogen ^^ j, f Glucose Glucose 1 phosr>hr;te

* ..

Glycerol Fatty Acid

it i t Fructose diuhosphnte 1 t ' lucose-»Phosphoglyceralrlehyd 00 Phosphoglycerate J, t I t Piso spho py ruva t e

Valine (F)

^Glucose 6 phosnnate

Cys4eine(R)

Cys^ine(H )

Alanine(R) (P)_____ Rerini e (R)

Oxaloacetate t Iv.late(E)

t

Furr; a r ate

X ^ Oil'll ^Pyruvate (T) I f A ce tv p.io s ph a.t e