A cytological and serological study of Spirillum serpens
by Curtis J Wilder
A THESIS Submitted to the Graduate Committee In partial fulfillment of the requirements for the
degree of Master of Science In Bacteriology
Montana State University
© Copyright by Curtis J Wilder (1941)
Abstract:
A cytological and serological study was made of the bacterial organism Spirillum serpens (Muller)
Winter, An effort was made to determine the way In which this species forms Its flagella and where the
flagella originate In the cell, A staining technic was developed by which certain polar granules could be
demonstrated In the cell while It was in the process of flagella formation, and also granules were
stained actually lying within the flagella. The flagella arise In close association with granular activity as
new structures with serological properties differing from the soma, The flagella have been observed to
be forming at different stages of cell division.
Serological studies using the precipitin reaction were made of this same species using three portions of
the cell material—the flagella, the soma, and the whole cells. From the results of this serological study
it is believed by the writer that there is possibly a different system or basic morphological pattern
between the soma and the flagella; that the flagella contain a much greater amount of precipitinogenic
material than the soma; and that the soma contain a substance capable of reacting with the
flagellar-precipitin in the immune serum. A CYTOLOGICAL AND SEROLOGICAL STUDY
OP SPIRILLUM SERPENS
by
CURTIS J . WILDER
A THESIS
S u b m itted t o th e G raduate Committee
in
p a r t i a l f u l f i l l m e n t o f th e r e q u ir e m e n ts
f o r th e d e g r e e o f
M aster o f S c ie n c e in B a c t e r io lo g y
at
Montana S t a t e C o lle g e
Approved:
In Charge o f Ma^or Work
[airm an , Examinin g CoTm^&tee
!airm an . G raduate Committee
Bozeman, Montana
J u n e, 1941
2
TABLE OP CONTENTS
Page
A b s t r a c t ................................................. ..
S
I n t r o d u c t io n .......................................
4
S e r o l o g i c a l S t u d y .................................
9
C y t o lo g lo a l M a te r ia l and Methods
18
G en eral D i s c u s s i o n .......... ..
23
Summery and C o n c l u s i o n s ..................
34
L it e r a t u r e C i t e d .................
36
3
ABSTRACT
A o y t o l o g l c a l and s e r o l o g i o n l s tu d y was made o f th e
b a c t e r i a l organ ism S p ir illu m se r p e n s (M u lle r ) w i n t e r . An
e f f o r t was made t o d e ter m in e th e way In w hich t h i s s p e c i e s
form s i t s f l a g e l l a and where th e f l a g e l l a o r i g i n a t e In th e
c e ll,
A s t a i n i n g t e c h n ic was d e v e lo p e d by w hich c e r t a i n p o la r
g r a n u le s c o u ld be d em o n stra ted In th e c e l l w h ile I t was In th e
p r o c e s s o f f l a g e l l a fo r m a tio n , and a l s o g r a n u le s w ere s t a in e d
a c t u a l l y l y i n g w it h in th e f l a g e l l a .
The f l a g e l l a a r i s e in
c l o s e a s s o c i a t i o n w ith g r a n u la r a c t i v i t y as new s t r u c t u r e s
w ith s e r o l o g i c a l p r o p e r t ie s d i f f e r i n g from th e som a,
^he
f l a g e l l a have b een o b serv ed t o be form in g a t d i f f e r e n t s t a g e s
o f c e l l d iv is io n .
S e r o l o g i c a l s t u d i e s u s in g th e p r e c i p i t i n r e a c t i o n were
made o f t h i s same s p e c i e s u s in g th r e e p o r tio n s o f th e c e l l
m a t e r ia l— th e f l a g e l l a , th e som a, and th e w h ole c e l l s .
From
th e r e s u l t s o f t h i s s e r o l o g i c a l stu d y i t la b e l ie v e d by th e
w r it e r t h a t th e r e i s p o s s i b l y a d i f f e r e n t sy stem or b a s ic
m o r p h o lo g ic a l p a t t e r n b etw een th e soma and th e f l a g e l l a ; t h a t
th e f l a g e l l a
c o n t a in a much g r e a t e r amount o f p r e c l p i t t n o g e n l c
m a t e r ia l than th e soma; and t h a t th e soma c o n ta in a su b sta n c e
c a p a b le o f r e a c t in g w ith th e f l a g e l l a r - p r e c i p i t i n
Immune serum .
In the
4
INTRODUCTION
'ork h a s b e n done bu t very few c o n c lu s io n s h ave been
r e a c h e d c o n c e r n in g th e fo rm a tio n o f b a c t e r i a l f l a g e l l a and
t h e i r c o n n e c tio n w ith th e c e l l .
Some w orkers b e l i e v e t h a t th e
f l a g e l l a a r e a c o n t in u a tio n o f th e e c to p la s m and o th e r s th a t
th e f l a g e l l a s p r in g from th e endoplasm or c y to p la sm and
e x te n d through th e o u te r w a l l .
That th e r e Ie an o u te r s t r u c t u r e has b een g e n e r a lly a c ­
c e p t e d , b u t I t s e x a c t c o m p o sitio n l a s t l l l unknown.
Mutld,
P o le v lt s k y and A nderson (1 9 4 1 ) by th e u se o f th e e le c t r o n
m icr o sc o p e have d em o n stra ted th e e x i s t e n c e o f a s o l i d c e l l
membrane, d i f f e r e n t i a t e d from an in n e r f l u i d or p o t e n t i a l l y
f l u i d p r o to p la sm f o r s e v e r a l r e p r e s e n t a t iv e b n c t r l a ; s t r e p t o ­
c o c c i , B a c i l l u s s u b t l l i a Cohn emend, Prazm ow skl, flC o llfo r m f1
and ty p h o id b a c i l l i .
They s t a t e a l s o t h a t th e In n er p ro to p la sm
may s h r in k away from th e c e l l membrane on d r y in g or may e sc a p e
In c y t o l l s l s , le a v in g th e membrane as a " g h o st" .
I t h a s b een
s u g g e s t e d by P o le v lt s k y (1 9 4 1 ) o f Mudd, P o le v lt s k y and Anderson
t h a t th e f l a g e l l a o f E b e r t h e lla typh oaa (Z op f) E e ld ln and
" co lifo rm " b a c t e r ia may be tu b u la r s t r u c t u r e s ; and fu r th e r
t h a t th e rh yth m ic c o n t r a c t io n o f th e p ro to p la sm In th e f l a g e l l a
c a u s in g p r e s s u r e ch a n g es in th e lumen o f such t u b u le s m ight
w e l l d eterm in e f l a g e l l a r m ovement.
T h is h y p o t h e s is s u g g e s ts
t h a t th e f l a g e l l a a r e o f b oth e c to p la s m and endo la sm ,
Tt h as
b een d em o n stra ted o u l t e c o n c l u s i v e l y , h ow ever, t h a t th e f l a g e l l a r
5
m a t e r ia l and th e so m a tic m a t e r ia l show a s e r o l o g i c a l d i f ­
fe r e n c e .
T h is s u g g e s t s th r e e p h a se s o f th e w h ole c e l l — endo­
p la sm , e c to p la s m and f l a g e l l a ,
L e lfa o n (1 9 3 0 ) p o in te d o u t t h a t M lgula (1 9 0 0 ) In h i s
"System d er B a c t e r le n n c o n clu d ed t h a t f l a g e l l a were ou tgrow th s
o f th e b a c t e r i a l e c to p la s m w hich seem ed t o be th e g e n e r a l
c o n c e p tio n a t t h a t t im e .
L e lfa o n worked w ith p r e p a r a tio n s
o f Eb. ty p h o sa and P r o te u s v u l g a r i s Houaer and s u g g e s te d t h a t ,
from th e ap p earan ce o f f l a g e l l a
on th e s e p r e p a r a t io n s , a
d i f f e r e n t c o n c lu s io n from t h a t o f &'lgula may he drawn.
He
went on t o sa y t h a t n e i t h e r o f t h e s e o r g a n ism s, r~b. typ h osa
and P r o te u s v u l g a r i s , s t a i n s u n ifo r m ly w ith th e f l a g e l l a
s ta in .
he f l & g e l l a , th e e c to p la s m ic so n s and a c e n tr a l
colum n are s t a in e d r e d .
The r e s t o f th e o r g a n ism , u n le s s a
c o u n t e r s t a in I s u s e d . I s c o l o r l e s s , and when I t I s u sed th e
r e s t o f th e organ ism ta k e s th e c o l o r o f th e c o u n t e r s t a in .
In a nu aber o f c a s e s th e f l a g e l l a w ere se e n t o o r i g i n a t e In
th e c e n t r a l colum n and o n ly r a r e ly d id a f la g e llu m o r ig i n a t e
b etw een th e c e n t r a l colum n and th e e c to p la s m ic z o n e .
he s t a t e d t h a t most f r e q u e n t ly th e f l a g e l l a
A lthough
seem t o o r ig i n a t e
on th e s u r f a c e , L e lf s o n a l s o p o in te d o u t from h i s d ata ob­
t a in e d w ith Eb. ty p h o sa e s p e c i a l l y . I t m ight be c o n clu d ed
t h a t b a c t e r i a l f l a g e l l a a r e n o t o f e c to p la s m ic o r i g i n bu t
o r ig i n a t e In th e en d op lasm , and p erh ap s In some d e f i n i t e
s t r u c t u r e w it h in th e en d op lasm .
6
He foun d In th e e x p e r im e n ts on th e e f f e c t o f a d d it io n
o f s t r o n g h y d r o c h lo r ic a c id t o th e f l a g e l l a s t a i n t h a t i t was
n o t ic e d t h a t when a c o n c e n tr a t io n o f 0 ,1 norm al was reach ed
th e f l a g e l l a w ould no lo n g e r s t a i n e v e n ly b u t a s « s e r i e s o f
e v e n ly sp a ced round d o t s .
But I f th e a c id c o n c e n tr a t io n was
in c r e a s e d s t i l l f u r t h e r th e f l a g e l l a d id n o t s t a i n a t a l l .
T h is g r a n u la r a p p earan ce o f th e f l a g e l l a was a l s o d em on strated
by th e a d d it io n o f o a r b o l f u c h a in t o a h a n g in g drop o f V ib r io s
(and o th e r organ ism s a s w e l l ) t h a t some f l a g e l l a can be se e n
— n o t s ta in e d s o l i d l y but as a s e r ie s o f d o te .
The p r o b a b i l i t y
w ould seem t o be from t h e i r s t a i n i n g b e h a v io r th t th e y are
n u c le a r in n a t u r e ,
He c o n clu d ed h i s e x p e r im e n ts by s t a t i n g
t h a t e v id e n c e la produ ced t o show t h a t f l a g e l l a may n o t be o f
e c t o p la s m ic o r i g i n , b u t may o r i g i n a t e somewhere in th e endo­
plasm and t h a t th e r e are some i n d ic a t io n s t h a t f l a g e l l a a re
n o t hom ogeneous b u t c o n ta in m asses o f n u c le a r m a t e r ia l d i s ­
t r ib u t e d a t r e g u la r i n t e r v a l s .
A ls o in agreem en t w ith th e th e o r y t h a t f l a g e l l a o r ig i n a t e
in th e endoplasm a r e T o p ley and
lls o n
r e f e r back t o Trenkmann ( 1 8 9 0 ) , B i l l s
( 1 9 3 7 ) , p age 9 4 , who
( 1 9 0 2 - 0 3 ) , Puhrmann (1 9 1 0 )
and Meyer ( 1 9 1 2 ) , who b e l i e v e t h a t th e c e n t r a l p o r t io n o f th e
f l a g e l l a r th r e a d p a s s e s through th e c e l l membrane and i s in
d i r e c t c o n n e c tio n w ith th e c y to p la sm ,
Puhrmann b e l i e v e s t h a t
th e p ro x im a l e x tr e m ity i s c o n n e c te d w ith a g r a n u le o f c h r o m a tin .
T ills would th en be a n a lo g o u s t o th e b l e n h a r o n la s t , f i r s t
7
d e s c r ib e d and named by
/ebber ( 1 8 9 7 ) , In h i s work on Zamla
and th e p o l l e n tu b e a p p a ra tu s o f G in kgo,
He s t a t e d :
ffT am
n o t %ware t h a t any d i s t i n g u i s h i n g name h as b een a n n lie d to
such an o r g a n , and I would h e r e s u g g e s t th e name b le p h a r o p la a t
t o d i s t i n g u i s h them from o th e r org a n s o f th e C e l l e "
'ebber
d e r iv e d th e word from tw o g r e e k words m eaning " e y e la sh " or
" c lllu m " ; and "form ed",
T op ley and W ilson p o in te d out t h a t
th e c o n c lu s io n t h a t th e f l a g e l l a r th r ea d i s in d i r e c t con ­
n e c t io n w ith th e c y to p la sm Is a d v e r s e ly c r i t i c i z e d by Z ettnow
(1 9 1 8 ).
Tn h i s work w ith th e fu n g i B la s t o c l R d l a , C otn er (1 9 3 0 )
fou n d t h a t th e c Iliu m I s i n s e r t e d on th e plasm a membrane in
a h i g h ly r e f r a c t i v e b o d y , th e b l e p h a r o p la s t , w h ich la d e f i n ­
i t e l y c o n n e c te d t o th e t i p o f th e n u c le u s by a s i n g l e th r e a d ­
l i k e c o n n e c tio n ,
th e c i l i a
He summarized t h i s work in c o n n e c tio n w ith
and s t a t e d t h a t th e z o o sp o r e s o f th e s p e c i e s s t u d ie d
a l l have a d e f i n i t e b a s a l g r a n u le ( b l e h a r o n la s t ) a t th e
in s e r t io n o f each c lllu m ,
T h is g r a n u le i s c o n n e c te d w ith
th e n u c le u s by a d e f i n i t e str a n d ( r h l z o p l a s t ) ,
fHie c i l i a
d e v e lo p a s o u tg ro w th s from th e r e g io n o f a b a s a l g r a n u le w h ich
h a s rea ch ed a p o s i t i o n n ea r th e plasm a membrane a s a r e s u l t
o f n u c le a r a c t i v i t y .
The b a s a l a n n a ra tu s i s alw a y s o f n u c le a r
o r i g i n and seems t o be c osm osed o f s e v e r a l c h r o m a tic b o d ie s ,
one o f w hich form s th e b a s a l g r a n u le n o rm a lly r e t a in e d or
drawn back I n to th e n u c le u s and u s u a l l y forms t h e t i p or
8
b e a k - lik e p a r t o f th e organ d u r in g z o o sp o r e a c t i v i t y ,
When
th e z o o s p o r e s o f e i t h e r swimming s ta g e come t o r e s t , th e
b a s a l g r a n u le or g r a n u le s , a s th e c a s e may b e , m ig r a te back
t o th e n u c le u s and a p p a r e n tly become p e r t o f t h a t organ .
In th e w r i t e r s i n v e s t i g a t i o n th e r e has been s t a in e d by
th e u s e o f a m o d ifie d C ^ s a r e s - G ilf a mordant and a h ig h d i l u t i o n
o f a m ix tu re o f b orax and t h i o n i n , a d e f i n i t e b a s a l g r a n u le
or group o f g r a n u le s J u st i n s id e th e e c t o r la s m ic membrane n t
th e b a se o f th e f l a g e l l a .
In a l l i n d ic a t io n s t h e s e g r a n u le s
appear t o be ch rom atin m a t e r i a l.
In o r d e r t o make a more c l e a r d i f f e r e n t i a t i o n betw een
th e f l a g e l l a and th e som a, a s e r i e s o f s e r o l o g i c a l t e s t s was
m ade.
The r e s u l t s o f t h i s stu d y s u b s t a n t i a t e th e e y t o l o g t e a l
work done on th e f l a g e l l a t e d c e l l s o f Sp, s e r p e n s .
9
SEROLOGICAL STUDT
Many w orkers h ave n o te d th e s n e c l f i o l t y o f a n t ig e n m a te r ia l
in th e f l a g e l l a o f b a c t e r ia d i f f e r i n g from t h a t In th e soma.
T h ese have b een d e s ig n a te d as th e f l a g e l l a r ffHM and th e som n tlo
"0" a n t i g e n s .
The f l a g e l l a r - s o m a t i e h y p o th e s is was advanced
by Omlth and Reagh (1 9 0 3 ) t o e x p la in th e a c t io n o f a g g l u t i n a t i n g
s e r a p re p a r ed from a m o t ile s t r a i n o f th e hog c h o le r a b a c i l l u s
and from i t s n o n -m o tlie v a r ia n t .
The serum f o r th e m o tile
s t r a i n c o n ta in e d a g g l u t i n i n s w hich were d em o n stra ted by ab­
s o r p t io n t e s t s ,
F lo c c u la r and g r a n u la r a g g l u t i n a t i o n r e s p e c ­
t i v e l y w ere produ ced by th e a n ti-s e r u m o f th e m o t ile s t r a i n
w h ile th e a n ti-s e r u m f o r th e n o n -m o tlle v a r ia n t ca u sed g r a n u la r
a g g l u t i n a t i o n o n ly .
The n o n -m o tlle s t r a i n was u n a b le to
a b so rb th e f l o c c u l a t i n g a g g l u t i n i n s o f th e serum fo r th e
m o t ile s t r a i n .
I t was th o u g h t by m ic r o s c o p ic a l e x a m in a tio n
t h a t th e t e x t u r a l d i f f e r e n c e b etw een f l o c c u l a r and g r a n u la r
clu m p in g was a s t r u c t u r a l one d ep en d en t on th e p r e s e n c e or
a b sen ce o f a fo u n d a tio n , presum ab ly a g g lu t in a t e d f l a g e l l a ,
O rcu tt (1 9 2 4 ) co n firm ed t h e s e r e s u l t s and c o n c lu d e d th a t th e
f l a g e l l a p o s s e s s e d an in d iv id u a l a n t i g e n i c p r o p e r ty ,
^he
a l s o d em on strated t h a t th e f l a g e l l a r s u s p e n s io n , when h e a te d
t o 7 0 ° C ., was no lo n g e r a g g lu t in a b le b u t r e t a in e d I t s a n t i ­
g e n ic q u a l i t i e s .
A f t e r s h a k in g , th e f l a g e l l a w ere d eta ch ed
from thq b o d ie s o f Rb, typhoaa by Vokota (1 9 2 5 ) and ln n o c u la te d
i n t o r a b b it s t o o b ta in an a p p r o p r ia te serum .
He d em on strated
10
t h a t th e clu m ping w hich d e v e !Ioperf In f ItBfTellar s u s p e n s io n s
when an a p p r o p r ia te serum was added was a c t u a l l y due t o th e
a g g r e g a t io n o f f l a g e l l a .
Y okota c o n clu d ed t h a t th e f l a g e l l a
1E&*. ty p h o se c o n ta in e d a s p e c i f i c a g g l u t i n a t i n g and ag»
g l u t ln o g e n lc p r o p e r ty w hich d i s t i n g u i s h e d them from th e body
o f t h a t o rg a n ism .
A rkw right (1 9 2 6 ) con firm ed t h e s e r e s u l t s
by sh ow in g by means o f h a n g in g drop p r e p a r a tio n s th a t a pure
so m a tic scrum a g g lu t in a t e d th e b o d ie s o f th e b a c i l l i w ith o u t
Im p a irin g t h e i r m o t i l i t y w h ile a pure f l a g e l l a r serum le d
p r im a r ily t o an Im m o b iliz a tio n o f th e o r g a n ism s.
F a d ley (1 9 2 7 )
s u g g e s t e d t h a t th e s e r o l o g i c a l d i f f e r e n c e s b etw een f l a g e l l a t e d
o rg a n ism s and t h e s e same organ ism s d e p r iv e d o f or la c k in g
t h e i r f l a g e l l a m igh t be e x p la in e d In term s o f s o l u b l e s p e c i f i c
s u b s ta n c e s w ith o u t any r e f e r e n c e t o m o r p h o lo g ic a l e le m e n ts .
The w r i t e r b e l i e v e s th e r e can p o s s i b l y be a m o r p h o lo g ic a l
e le m e n t e n t e r in g I n to t h i s d i f f e r e n c e w hich i s e x p la in e d l a t e r ,
C r a lg le (1 9 3 1 ) a s c e r t a in e d t h a t th e nF n a n t ig e n o f ? e ll
and P e l l x (1 9 1 7 ) I s a s s o c i a t e d w ith th e f l a g e l l a o f an organism
and summarized th e r e s u l t s a s f o l lo w s !
H a n tig e n
P r e s e n t o n ly In f l a g e l l a t e d
o r g a n ism s.
R e s p o n s ib le f o r l a r g e - f l a k i n g
a g g lu tin a tio n .
H eat l a b i l e .
L a b ilo fcr o p lc a g g l u t i n i n Is
n o t Im paired by d e g r e e o f
h e a t in g w hich d e s t r o y s s t a b llo tr o p lc a g g lu tin in .
P ls g e lla
R e sp o n s ib le f o r f l o c o u l a r
a g g lu tin a tio n .
‘t hen h e a te d t o 7 5 ° c . are no
lo n g e r a g g lu t ln a b le or demons­
t r a b le m i c r o s c o p i c a l l y ,
P lo g e lla r a g g lu tin in Is not
Im paired by d e g r e e o f h e a t ­
in g w hich d e s t r o y s so m a tic
a g g lu tin in .
11
C r a lg le s t a t e d t h a t t h i s e v id e n c e does n o t n e c e s s a r i l y
mean t h a t f l a g e l l a c o n s i s t e n t i r e l y o f "H" a n t ig e n ,
G ibson (1 9 3 2 ) a g r e e s w ith th e many w orkers in t h i s f i e l d
t h a t th e c l e a r su p e r n a ta n t l i q u i d o f Vl b r lq comrna (V o h ro e ter )
may be em ployed as an "H" a n tig e n In a g g l u t i n a t i o n r e a c t i o n s .
V a rio u s m o t ile organ ism s were t r e a t e d t o o b tn ln su ch f l a g e l ­
l a r s u s p e n s io n s .
The r e s u l t s were q u it e d e f i n i t e , l a r g e ,
f l o c c u l e n t m asses a p r e a r in g r a p id ly in such t e s t s ,
Xt was
found t h a t su ch ap e a r a n e e s c o u ld be produced in a l l c a s e s
where a sam ple o f serum r e a c te d In e v en m o d e ra te ly h ig h d i l u t i o n
w ith an u n tr e a te d s u s p e n s io n o f a m o t ile o r g a n ism ,
Tt i s now
g e n e r a l ly a c c e p te d t h a t th e "R" a n t ig e n i s found o n ly in
m o t ile organ ism s and la a s s o c i a t e d w ith th e f l a g e l l a ,
In m ost o f th e work c a r r ie d out unon f l a g e l l a r and so m a tic
a n t i g e n s , th e a g g l u t i n a t i o n r e a c t i o n was u sed as a m easure o f
th e s n r o l o g ' c a l s p e c i f i c i t y o f th e a n t ig e n and I t s t i t e r .
th e a u th o r ’ s work w ith th e f l a g e l l a t e d
Tn
s e m e n s . th e
p r e c i p i t i n r e a c t i o n was u sed w ith e x c e l l e n t r e s u l t s .
M a te r ia ls and Ve th o d a , - - Sn. s e m e n s was grown on d e x tr o s e
agar s l a n t s and h a r v e s te d tw e n ty -fo u r hou rs a f t e r ln n o o u la t lo n .
A s u s p e n s io n o f th e o rgan ism s was made in p h y s i o l o g i c a l s a l t
s o l u t i o n and an a l iq u o t n a r t was s e t a s id e t o be in n o c u la te d
I n to th e r a b b it a s w hole c e i l s .
The rem a in in g s u s p e n s io n was
p la c e d In a s t e r i l e b o t t l e w ith s t e r i l e
g l a s s b ead s ^nd shaken
by hand approxim t e l y f i v e t o t e n m in u te s .
I t was th e n c e n t r l -
12
fu g e d In a m ilI a g g l u t i n a t i o n t u b e s , th e c l e a r l l c u l d c o n t a in in g
th e su sp en d ed f l a g e l l a was drawn o f f by a s t e r i l e n i p e t t e and
p la c e d In a s e p a r a te tu b e .
The r em a in in g some p o r t io n o f th e
c e l l s was r e -su sp e n d e d w ith p h y s i o l o g i c a l s a l t s o l u t i o n up to
th e o r i g i n a l mark and u sed a s a so m a tic v a c c in e .
The r a b b it s
were ln n o c u la te d l n t r a p e r l t o n e a l l y a t f i v e day i n t e r v a l s f o r
se v e n ! m o d u la t i o n s and th en b le d a s e p t Ic a ll'" from th e h e a r t s i x
d ays a f t e r th e l a s t ln n o o u la t Io n ,
The serum was s e p a r a te d from
th e b lo o d c e l l s , p la c e d in s t e r i l e tu b e s and s to r e d in th e
r e fr ig e r a to r .
The d e t a i l s o f th e r a b b it Im m unization are E lven in
T ab le I , w hich shows th e number o f in n o c u ln tlo n s and th e
amount o f in n o cv lu m .
The b a c t e r i a l s u s p e n s io n s t o be t e s t e d a g a in s t th e se r a
were p rep a red as above b u t th e b a c t e r i a l co u n t was 925 m i l l i o n
per c o ,
A f t e r s e p a r a t in g th e th r e e a n t lg e n s --w h o le c e l l s ,
som a, and f l a g e l l a - - t h e s u s p e n s io n s o f each were fr o z e n q u ic k ly
by p la c in g th e tu b es c o n t a in in g th e s u s p e n s io n s i n t o a m ix tu re
o f I c e and NaCl and th e n thawed im m e d ia te ly .
The p roced u re
was r e p e a te d f i v e tim e s to e n a b le th e s o l i d c e l l u l a r and f l a ­
g e l l a r sy ste m s t o be l l o u l f l e d
or t o l i b e r a t e th e p r o t e in
sy ste m s from th e s o l i d I n to s o l u t i o n w hich was n e c e s s a r y fo r
th e p r e c i p i t i n r e a c t i o n .
I t would he I n t e r e s t i n g t o e x p l a in a t t h i s t "me t h a t
a f t e r th e fr e e z in g -th a w in g o r o c e s s
l u s t d e s c r ib e d , th e f I a g e l o r
IS
Tnble I
S u sp e n sio n o f
o r g a n ism s.
Whole
c e lls
Humber o f Trm ooulntI one and
Amount o f Innoculum .
I.
s,
3.
4.
5.
6.
7.
Soma
minus
fla g e lla
I.
a.
3.
4.
5.
6,
7.
F la g e lla
I.
a.
3.
4.
5.
6 .
7,
0 .3
0 ,3
0 .6
0 .6
1 ,0
1 .5
1 .4
, o f 200 m i l l i o n p er
tf
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it
cc.
H
it
it
cc.
c c , o f 725 m i ll i o n p er
n
«
it
CO.
c c , o f eon m i l l i o n p er
it
»
f?
cc.
0 .3
0 .3
0 .6
3 .2
1 .7
2 .4
2 .6
c c , o f 200 m i ll i o n p er
W
it
cc.
H
M
n
cc.
CO,
o f 725 m ill I on p er
tt
i*
if
CO.
c c . o f 800 m i l l i o n p e r
Ii
n
H
CC ,
Ce
1 .0 C O , o f 200 m i l l i o n p er
«
n
it
1 .0 c c ,
n
n
H
1 .9 C O .
2 .2 c c . o f 725 m i l l i o n p er
B
H
ft
2 .5 c c .
3 .0 C O . o f 800 m i l l i o n p er
M
it
IT
3 .0 C O ,
CO
CC
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CC
CC
CC
CC
CC
and w hole c e l l s s u s p e n s io n d e v e lo p e d a d I n s t i n c t y e llo w e a s t ,
w h ile th e so m a tic s u s p e n s io n rem ained p u r e -w h ite in c o l o r .
T h is may be I n t e r p r e te d as an i n d ic a t io n o f a d i s t i n c t d i f ­
f e r e n c e b etw een th e soma and th e f l a g e l l a .
T h is c o l nr d i f ­
f e r e n c e m ight s Imply be due t o p ig m e n ta tio n c h a n g e s ,
The r e s u l t s o f th e n r e c l n i t l n r e a c t io n s a r e g iv e n In
T a b le I I ,
In th e i n d ic a t e d d i l u t i o n s th e f i r s t d i l u t i o n ,
1 /1 0 0 0 , c o n ta in e d 9 2 5 ,0 0 0 c e l l s p e r c c , or In th e c a s e o f th e
f l a g e l l a th e f i r s t d i l u t i o n c o n ta in e d th e f l a g e l l a
from 9 2 5 ,0 0 0 w hole c e l l s per c c ,
se p a r a te d
The c e l l s *md th e f l a g e l l a
14
h a v e presum ably b een n u rtu red f o llo w in g th e f r e e 2 I n g - thaw in g
a c t i o n and onn nor be u sed in th e n r e c l n i t i n r e a c t i o n .
The r e a c t i o n s w ere rea d in sm a ll p r e c i p i t i n t u b e s .
A p nroxlm ateIy 0 ,1 0 o e . o f serum wsts n la c e d 1Ti th e b ottom o f
th e tu b e by means o f a C a p illa r y p i p e t t e and th e aaino amount
o f d i l u t e d a n t ig e n was th en la y e r e d above th e sem en,
Cnre
was ta k en t o o b ta in a p r e c i s e l i n e o f c o n ta c t b etw een th e two
flu id s .
The r e a c t i o n s w ere read a t th e end o f th e f i r s t and
seco n d h o u rs a f t ^ r b e in g s e t u p .
In r e c o r d in g th e r e a c t i o n s , a p r o fu s e p r e c i p i t a t e show ing
c o n s id e r a b le depth a t th e Iu n c tto n o f th e immune serum and th e
a n t ig e n e x t r a c t was read ns a f o u r - p lu s r e a c t io n
(+ + + + ), a
r l n r w ith Ien e d ep th was rend a s n t h r e e - r l u s r e a c t i o n (+ + + ),
th e l i g h t e r b u t d e f i n i t e r in g s a s tw o -n in e r e a c t i o n s (+ + ),
and a r e a c t i o n In w h ich no d e f i n i t e r in g was form ed h u t In
w hich a s l i g h t p r e c i p i t a t e w^s v i s i b l e was read am a o r e - p iu s
r e a c tio n ( + ) .
By r e f e r r i n g t o T ab le I I , w hich showr th e r e a c t io n s b e ­
tw een th e immune s e r a and a n t i g e n s , it, w i l l be n o te d th a t th e
s t r o n g r e a c t i o n s ta k e p la c e in th e h ig h d i l u t i o n s w ith th e
P (fla g e lla r )
Immune serum .
I t i s o b v io u s t h a t th e serum
from th e r a b b it immunised w ith th e f l a g e l l a r s u s p e n s io n has
a v e r y h ig h t i t e r — much h ig h e r th a n th e serum from th e r a b b it
Immunized a g a in s t th e soma and th e one Immunized a g a in s t th e
w hole c e l l s .
There i s a d e f i n i t e t i t e r in th e l a s t two
15
T ab le I t
P r e c i p i t i n R e a c tio n s
D i l u t i o n s o f E x tr a c t
S era A n t.
e x t.
F
P
V
V
4 0 .0 0 0 5 0 .0 0 0
V
i/
SO. 000 100
+++
F
S
S
S
W
F
++
++
++++
++++
4--M-44-4-4-4-
+++
444
+++
4-++
++
+++
4
44
+-H-
+++
+++
++
+4
+
H ++
4-44-4-4-
+++
+++
H
44
++
+++
+4-4-
+++
++
44
44
+++
+++
+++
+4
++
+ ++
+■*-
4-
+
-
+
++
++
++
++
++
«•>
+
4
•
+
+>
++
++
+
+++
++
4
+
4
-
-
+
+
4
mm
+
+
-
S
W
+
+
+
+
+
W
F
♦
•*
+
+
4-
W
+
+
-
+
4+
4-
+
4-
S
+
W
+
++
++
S
W
V
4*4*4*
+++
F
V
I/
I/
1000 5 0 0 0 1 0 .0 0 0 2 0 .0 0 0
-H-
-H
+
«•
4
4
+
*
+
+
+
+
m
4*
In b o th th e serum and th e a n t ig e n e x t r a c t :
8 s Soma, and W » s h o le c e l l s ,
P s F la g e lla ,
16
m en tion ed r a b b it s s e r a a s th e r e la a s l i g h t r e a c t i o n betw een
th e soma a n t ig e n e x t r a c t and I t s hom ologous lrm in e serum and
a l s o b etw een th e w h ole c e l l a n t ig e n e x t r a c t and I t e hom ologous
Immune serum .
But In no cane do t h e s e r e a c t 'o n e reach am
h ig h a d i l u t i o n a s t h a t a t t a in e d by th e f l a g e l l a
immune serum .
There seem s t o be a d i s t i n c t s e p a r a te p r e c ip lt in o g e n .ie
s u b s ta n c e in th e f l a g e l l a
a h ig h t i t e r .
t h a t c a u s e s th e r a b b it t o nroduce
There must a l s o o c cu r In th e soma o f th e
organism a c o r r e sp o n d in g s u b s ta n c e t h a t , a lth o u g h i t does
n o t have th e power t o i n c i t e
in th e r a b b it th e n r o d u c tlo n o f
a h ig h p r e c i p i t i n t i t e r , d oes r e a c t s t r o n g ly w ith th e f l n g e l l a r
p r e c ip itin ,
Tn o th e r w ord s, th e r e s u l t s from t h i s e x p erim en t
show t h a t th e soma o f Bp, serp en o c o n ta in s a su b s ta n c e th a t
can n o t i n c i t e p r e c i p i t i n fo r m a tio n b u t r e t a i n s i t s newer
t o r e s e t w ith th e p r e c i p i t i n w hich I s p r e s e n t from th e a c t io n
o f th e f l a g e l l a r p r e c i p i t i n o g e n .
T h is p r e c i p l t l n o g e n l c s u b s ta n c e o f th e f l a g e l l a c o u ld
p o s s i b l y be l o c a t e d In th e plasma membrane or in a th in mem*,
brane t h a t s im u la t e s a c a p s u le t h a t may c o m p le te ly surround
th e f l a g e l l a and th e soma o f th e o r g a n ism s.
T h is su b sta n c e
may be compared t o th e t y p e - s p e c i f i c su b sta n c e in th e c a p s u le
o f th e pneum ococcus, b u t w h ile th e c a p s u le o f th e Tmeumococcus
i s v i s i b l e under c e r t a i n c ir c u m s ta n c e s , th e t h in c a p s u l e - l l k e
f i lm su rr o u n d in g th e f l a g e l l a t e d organism i s u n d e te c ta b le by
o r d in a r y m ic r o s c o p ic a l m eans.
h e n th e f l a g e l l a a r e form ed.
17
t h i s f ilm c o n t a in in g th e s p e c i f i c a n t ig e n ic m a t e r ia l c o v e r in g
th e soma w ould a l s o c o v e r th e f l a g e l l a .
g r e a t e r amount o f s u r fa c e on th e f l a g e l l a
B eca u se th e r e i s a
th an on th e soma
o f th e o rg a n ism , a s e r o l o g i c a l t e s t would ^ e v e a l th e p r e se n c e
o f t h i s a n t i g e n i c m a t e r ia l w ith th e f l a g e l l a much more s t r o n g ly
th a n w ith th e som a.
T h is v ie w p o in t e x p la in s th e s e r o l o g i c a l
d i f f e r e n c e b etw een th e f l s g e l l a and th e som a, n o t as m
q u a l i t a t i v e t e s t b u t p u r e ly a s a q u a n t i t a t i v e t e s t ca u sed by
th e d i f f e r e n c e In th e amount o f s u r f a c e o f th e f l a g e l l a and
th e som a.
T h is may e x p la in th e h ig h p r e o l n l t t n r e a c t io n shown
In T a b le I I b etw een th e fla g e lla r -Im m u n e serum and th e w hole
c e l l , soma, and f l a g e l l a a n t i g e n s .
There I e a much g r e a t e r
p e r c e n ta g e o f s u r fa c e In p r o p o r tio n t o th e c y to p la s m ic m a te r ia l
In th e f l a g e l l a and h en ce a g r e a t e r p e r c e n ta g e o f f l a g e l l a r 1
o r s u r f a c e a n t ig e n b r in g in g ab ou t a h ig h e r d e g r ee o f s p e c i f i c
Immunity In th e r a b b it th an th e soma a n tig e n I s a b le t o a t t a i n .
If?
CYTOLOGICAL HATERIAL AHD METHODS
The e t r e l n o f Sp. se r p e n s s t u d ie d wnc o b ta in e d from th e
s t o c k c u lt u r e c a r r ie d by Montana S t a t e C o lle g e f o r u se In
b a c t e r i o lo g y c o u r s e s .
nTie c u lt u r e was t r a n s f e r r e d e v er y day
or. d e x t r o s e a g a r s l a n t s — O.Z< b e e f e x t r a c t , 0 ,5 ^ p e p to n e ,
1 ,2 $ a g a r , and I , O'f d e x t r o s e .
Tt was found t h a t t h i s organism
grew v e r y f a s t and p l e n t i f u l l y a t room te m p e r a tu r e .
P r e v io u s t o a c t u a l s t a i n i n g , th e b a c t e r ia w ere tak en
d i r e c t l y from th e s l a n t and su sp en d ed In a drop o f ^ a te r on
a c le m
s lid e .
The drop o f s u s p e n s io n was th e n sp rea d e v e n ly
on th e s l i d e and a llo w e d t o e v a p o r a te t o d r y n e ss b e fo r e p ro­
c e e d in g w ith th e s t a i n i n g r e a c t i o n s .
trtM s method o f p r e p a r in g
th e smear was found t o be s u p e r io r t o th e u s e o f osm lc a c id
fum es f o r k i l l i n g th e b a c t e r ia b e fo r e d r y in g .
‘Then th e s e
f vmes were u se d th e c e l l s were s t a i n e d l i g h t e r and w ith l e e s
d iffe r e n tia tio n .
In o r d e r t o s e c u r e c le a n m icr o sc o p e s l i d e s f o r th e f l a g e l l a
s t a i n s , new s l i d e s w ere a I low ed t o rem ain in c le a n in g s o l u t i o n
(co m m ercia l s u l f u r i c a c id s a t u r a t e d w ith p o ta s siu m dleh rom M e)
f o r f i v e t o te n m in u te s , r in s e d th o r o u g h ly In f r e s h ta p w a ter
and f i n a l l y p la c e d In a b e a k e r o f d i s t i l l e d w a ter c o v er ed t o
keep out d u s t .
Im m ed iately b e fo r e u s in g n s l i d e i t was ta k en
from th e b ea k er o f d i s t i l l e d w a te r , washed w ith d i s t i l l e d w a te r
and h e ld above th e flam e o f a bunaen bu rn er u n t i l th e w ater
had c o m p le te ly e v a p o r a te d .
To a c co m p lish t h i s , and fu r th e r
19
c le a n th e s l i d e i t was h e a te d q u i t e h o t .
C a u tio n was tak en
t o be su r e th e s l i d e had c o o le d b e fo r e p r o c e e d in g w ith th e
s ta in in g r e a c tio n s .
S e v e r a l d i f f e r e n t m ordants were t r i e d f o r th e s t a i n i n g
o f th e f l a g e l l a o f Sp. se r p e n s b u t o n ly o n e , C a a a r e s - C ll*s
m ord ant, Z in s s e r and B ayne-Jonea ( 1 9 3 7 ) , was foun d t o be
c o m p le te ly s a t i s f a c t o r y b o th In e a s e o f h a n d lin g and r e s u l t s
o b t a in e d .
M ordants are v e r y u n s t a b le m ix tu r e s and a s
'r ig h t
(1 9 2 8 ) I n d ic a t e d , more c o n s i s t e n t r e e u l t s would p ro b a b ly be
o b ta in e d I f th e y w ere o f s t a b le c o m p o sitio n and more was known
o f th e f a c t o r s i n f l u e n c in g th e mechanism o f t h e i r a c t i o n .
Many o f th e m ordants change r a p i d l y , b o th c h e m ic a lly , and
p h y s i c a l l y a f t e r p r e p a r a tio n .
The w r it e r u sed C a a a r e s - C ll1*
mordant d u r in g th e c o u r se o f th e I n v e s t i g a t i o n o f th e f l a g e l l a
and when e x c e l l e n t r e s u l t s had b een o b ta in e d I t was d is c o v e r e d
t h a t t h e s e i n v e s t i g a t i o n s a g r ee d w ith '/r ig h t, f o r he s t a t e d :
" P r e p a r a tio n s were m ordanted a t 2 5 ° C ., 3 0 c C ., 5 5 ° C ,, and
5 7 ° C.
T em peratures above 3 0 ° C. In c r e a se d th e r a t e o f
p r e c i p i t a t i o n and c a u se d g r e a t c l o t s t o form on th e s l i d e s ,
e v e n when In a v e r t i c a l p o s i t i o n .
The b e s t r e s u l t s were
o b ta in e d a t 2 6 ° C, w ith th e C a s a r e s -R ll m ordant,
L o e f f l e r 1S
mordant gave good r e s u l t s a t h ig h e r te m p e r a t u r e s ,”
The scheme u se d by th e w r it e r f o r th e C o s n r e s - C ll’ a
m ordant I s g iv e n b e Iowt
20
T annic a c i d ........................................................................
Aluminum c h l o r i d e ..........................
Z inc c h l o r i d e ....................................................................
R o s a n llin e h y d r o c h lo r id e ................
A lc o h o l f S 0 < ) . , . . . ..................................................
1 0 .0
1 8 .0
1 0 .0
1 .5
4 0 .0
gres.
trma.
fm s .
gms.
oc.
The s o l i d s were d i s s o l v e d in th e a lc o h o l by t r i t u r a t i o n
in a m o r ta r , a d d in g 10 c o , o f th e a lc o h o l f i r s t , and th en th e
r e s t s lo w ly .
I t was found t h a t t h i s mordant a c te d b e s t a f t e r
a llo w in g i t t o sta n d a s t h i s a l c o h o l i c s o l u t i o n in a c lo s e d
v e s s e l f o r ab ou t two w eeks and th e n u s e d .
T h is mordant
worked s a t i s f a c t o r i l y f o r a p e r io d o f ab ou t two w e e k s.
Im­
m e d ia te ly b e fo r e u s i n g , one p a r t o f th e mordant was mixed
w ith a b o u t fo u r p a r t s o f w a te r , sh ak en or s t i r r e d w e ll and
th e n f i l t e r e d th rou gh f i l t e r pap er d i r e c t l y o n to th e f r e s h l y
d r ie d b a c t e r i a l s u s p e n s io n .
In some p r o c e s s e s o f b a c t e r i a l s t a i n i n g th e o b j e c t h a s
b een t o s t a i n h e a v i l y so t h a t one g e t s # c l e a r p r o f i l e or
s i l h o u e t t e o f th e b a c t e r i a l c e l l w hich a id s in th e r e c o g n it io n
o f th e s p e c i e s .
In th e w r i t e r ' s work th e p r i n c i p l e o f u n d er-
s t a i n i n g m ost s t r i k i n g l y i l l u s t r a t e d by C otner (1 9 3 0 ) was u s e d .
In t h i s way th e b a c t e r i a l c e l l la s t a in e d a v e r y l i g h t shade
o f th e c o lo r o f th e s t a i n u sed and th e n , when n c o u n t e r s t a in
I s ad d ed . I t shows a h ig h d eg r ee o f d i f f e r e n t i a t i o n .
In f o l lo w in g th e d evelop m en t o f th e f l a g e l l a s e v e r a l
s e r i e s o f s t a i n s were made from th e same c u lt u r e t o d eterm in e
th e m ost s u i t a b l e tim e f o r th e o b s e r v a tio n o f th e a c tu a l
p r o c e s s o f fo r m a tio n .
I t was n o t ic e d from t h i s , t h a t th e
21
o ld e r c u l t u r e s (1 8 -2 4 h o u r s ) n eed ed an in te r m e d ia r y s t a i n t o
d i f f e r e n t i a t e th e g r a n u la r , v a c u o la te d o ld e r c e l l s from th e
y o u n g er o n e s .
The s t a i n u sed was a m o d if ic a t io n o f th e c a r b o l
f u c h s in ( b a s i c ) s t a i n .
F o llo w in g t h i s s t a i n a h i g h ly d ilu t e d
m ix tu re o f t h io n in end b orax was u se d to b r in g out. th e g r a n u le s
o f th e SOEto and f l a g e l l a .
In th e you n ger c u l t u r e s (6 -7 h o u r s)
t h i s in te r m e d ia r y s t a i n o f c a r b o l f u c h s in was n o t needed and
o n ly o b scu red th e c l a r i t y o f th e s t a i n .
The f o l lo w in g form ula
was u sed f o r th e c a r b o l f u c h s in s
S o lu t io n A
A lo c h o l ( 9 5 t ) ........................
B a s ic fu c h s i n ............................. ..
3 0 ,0
oc.
0 0 .0 0 9 gms,
S o lu t io n B
D i s t i l l e d w a te r .............................. 2 8 5 .0
P h e n o l . , . , , , . . , . . . . . . . , . . . . . 1 5 ,0
cc.
gms.
h e n th e s o l i d s w ere d i s s o l v e d th e s o l u t i o n s w ere m’xed and
u sed from a d r o p p in g - b o t t le ,
Ih e t h io n in - b o r a x s o l u t i o n c o n s is t e d o f 0 ,1 < t h io n in
p lu s 0 ,5 $ b o ra x in d i s t i l l e d w a te r and t h i s s o l u t i o n d i l u t e d
1 :1 0 0 f o r th e f i r s t a p p l i c a t i o n .
In a t y p i c a l f l a g e l l a s t a i n o f a 2 0 -3 0 hou r c u lt u r e th e
f o llo w in g tim e scheme was u sed :
C a s a r e s - G il1S m o r d a n t.............................
3 m in u te s .
M o d ified C arbol f u c h a in .................................... 12 m in u te s .
T h io n in (+ b o ra x ) d i l u t e d 1 : 1 0 0 .................
I m in u te .
T h io n in (+ b o r a x ) u n d ilu t e d ........................... 10 s e c o n d s .
^ash w ith d i s t i l l e d w a te r .
Som etim es th e s t a i n e d s l i d e s were c le a r e d in pure c lo v e o i l
f o r 1 6 -2 4 h o u rs b u t t h i s was found t o make no g r e a t d if f e r e n c e
and was se e n aban don ed .
22
In th e you n ger c u l t u r e s o f s i x t o sev en h o u rs th e f o l ­
lo w in g tim e scheme was u se d !
G a sa r esw G lll S m ordant............................. ..
5 .5 m in u te s ,
T h lo n ln (+ b o r a x ) d i l u t e d 1 :1 0 0 ............. 10 m!n o t e s ,
ash w ith d i s t i l l e d w a te r .
T h is l a s t sc hema worked v e r y w e l l and a s can be s e e n . Is g r e a t l y
s im p lifie d .
S3
GENERAL DISCUSSION
Hi© w r i t e r b e l i e v e s t h a t In some r e s p e c t s a com parison
b etw een th e ftm g l and b a c t e r ia m igh t be s e t up In r eg a rd to
th e ir c i l i a
fo r m a tio n .
The r e s u l t s o f t h i s work d e a lin g w ith
th e fo r m a tio n o f th e f l a g e l l a
o f 3 n . se r p e n s i s in many d e t a i l s
In agreem ent w ith C o tn e r 1a (1 9 3 0 ) work on th e fo r m a tio n o f
c ilia
in th e O om ycetes, and in agreem ent w ith Euhrmann (1 9 1 0 )
ard L e lf s o n ( 1 9 3 0 ) .
C otn er (1 9 3 0 a , 1930b , 1 930c) made a c y t © lo g ic a l stu d y o f
th e z o o sp o r e s o f m yxom yoetea, B l a s t o c la d la and O om ycetes.
In h i s work w ith H e t lc u la r ia ly c o p e r d o n B u l l . he found t h a t
th e o l l l a - f o r m i n g organ in th e z o o sp o r e in com posefi o f s e v e r a l
g r a n u le s and a s th e c lllu m m atures t h e s e g r a n u le s m ig ra te
b ack toward th e n u c le u s and t h a t t h i s com plex o f g r a n u le s ,
w hich a p p ea rs a s a s i n g l e la r g e g r a n u le a t th e b a se o f th e
c illu m o f th e m ature myxomycete z o o s p o r e , i s o f c o n s id e r a b le
im p o r ta n c e ,
He f u r t h e r s t a t e d :
"In th e z o o s p o r e s , e s p e c i a l l y
o f th e seco n d swimming s t a g e o f s p e c i e s o f A c h ly a . Aphanom yces.
and S a p r o le g n ia . th e w r i t e r h a s show n, h o w ev er, t h a t b oth
p o l e s o f th e n u c le u s , w ith t h e i r a tta c h e d or in c lu d e d g r a n u le s ,
approach v e r y n ea r t o or a c t u a l l y to u ch th e plasm a membrane $
th e y may th e n w ithdraw from th e membrane l e a v in g th e r e one or
more g r a n u le s ,
At p o i n t s n ear t h e s e g r a n u le s we f in d th e
c i l i a a p p e a r in g , f i r s t a s p s e u d o p o d -lik e p r o j e c t i o n s , which
l a t e r a r e m atured I n to a c t i v e c i l i a .
The q u e s t io n now a r i s e s .
24
a re t h e s e b a s a l g r a n u le s , w hich seem t o c o n t r o l t h e d evelop m en t
of c ilia
in th e z o o s p o r e s o f th e Ocmiycetes, hom ologous w ith
th e b a s a l a p p a ra tu s o f th e p r o t i s t a ami th o s e found In th e
a p e r m a to g e n sls o f some a n im a ls and p l a n t s ? ”
C otn er g o e s on f u r t h e r t o sa y t h a t I t seem s l i k e l y t h a t
th e b a s a l a p p a ra tu s in t h e s e fu n g i Ie v e r y d e f i n i t e l y o f
n u c le a r o r i g i n .
In th e secon d swimming s tn g e o f t h e s e fu n g i
I f h a s b^en shown t h a t t h e s e g r a n u le s , o f t e n t h r e e in number
f o r each c l l l u m , a r e f i r s t found o c cu p y in g p o s i t i o n s n ear th e
p o le s o f th e c r e s c e n t-s h a p e d n u c le a r a p p a ra tu s a s th e sp o re
prepares fo r c i l i a
fo r m a tio n .
The g r a n u le s may, h ow ever,
under c e r t a i n c o n d it io n s rem ain s e p a r a te from th e n u c le u s and
h ave co m p lete c o n t r o l o f th e d evelop m en t and f u n c t io n o f one
c iliu m i f th e g r a n u le s a t one p o le o f th e n u c le u s become
c o m p le te ly se p a r a te d from th e n u c le u s e a r ly in th e p r o c e s s o f
c ilia
fo r m a tio n , b u t rem ain In c l o s e p r o x im ity w ith each o t h e r .
In r e f e r e n c e t o th e seco n d swimming s t a r e o f A chlya
c o n s pI c u a . Aphanomyoes e u t e i c h e s . and A ap rolegrila monica v a r ,
g lo m e r a te C otn er s t a t e d :
" I t I s a p p a r e n t, th e n , t h a t even
th ou gh t h e s e b a s a l g r a n u le s a r e shown t o be o f n u c le a r o r ig i n
th e y may d e v e lo p and c o n t r o l c i l i a
a fter ,
in so f a r a s one can
s e e , th e y h ave b een c o m p le te ly s e p a r a te d from th e n u c le u s .
T h is would seem t o show t h a t th e o ’ I l a o f th e s p o r e s in t h e s e
f u n g i may have t h e i r o r i g i n and may be c o n t r o l l e d by d i f f e r ­
e n t i a t e d p a r ts o f th e n u c le a r m a t e r ia l and n o t by th e e n t i r e
25
o r g a n ."
At th e tim e o f e t a ln tn g th e aoectm en o f Sn. e er n e n s
shown In P i g . I , th e I n d iv id u a l c e l l was In th e s t a g e o f
d ev elo p m en t In w hich i t
I s j u s t b e g in n in g t o g a th e r a n u c le a r
or a t l e a s t a c o l l e c t i o n o f g r a n u la r m a t e r ia l a t one n o le ,
In a lm o st a l l c a s e s t h e s e g r a n u le s seem ed t o be a c o l l e c t i o n
o f s m a lle r on es o f d i f f e r e n t a Iz e and n o t one huge g r a n u le .
The end o f th e c e l l I s p o in te d s l i g h t l y .
T hether or n o t t h l e
I s a c h a r a c t e r i s t i c fo r m a tio n o f th e a c tu a l b e g in n in g o f th e
th r e a d in g ou t o f th e f la g e llu m i s hard to d e te r m in e , bu t
n e v e r t h e l e s s th e cy to p la sm and th e plasm a membrane a t th e end
o f th e c e l l a re n o t rounded sm o o th ly .
The sm a ll dark g r a n u le
a t th e o p p o s it e end may e a s i l y be th e f i r s t o f a groun to
a p p e a r , p r e lim in a r y t o th e f l a t r e ll a fo r m a tio n .
The w r ite r
b e l i e v e s t h a t t h i s s i n g l e g r a n u le or goup o f g r a n u le s i s
com parable t o th e b le p h a r o n la s t in th e c i l i a
fo rm a tio n o f
c e r t a i n fu n g i and o th e r m o tile p la n t and anim al c e l l s .
S in c e t h e s e g r a n u le s are s t a i n e d by nu cle& r s t a i n s .
I t seem s r e a s o n a b le t o I n t e r p r e t them a s b e in g composed o f
n u c le a r m a t e r i a l.
I t seems h ig h ly im probable t h a t th e s e
g r a n u le s a t th e b a se o f th e f l a g e l l a c o u ld p o s s i b l y have
a r is e n as th e r e s u l t o f c y to p la s m ic a c t i v i t y .
I f th e s e
g r a n u le s had d e v e lo p e d a s a r e s u l t in s te a d o f a s th e c a u se
o f f l a g e l l a fo r m a tio n . I t seem s l i k e l y th a t th e r e would be
a g r e a t e r c o n c e n tr a t io n o f g r a n u le s a f t ^ r th e f l a g e l l a fo r m a tio n
26
and n o t b e f o r e .
In P ig , I th e la r g e group o f g r a n u le s has
g a th e r e d p r e lim in a r y t o f l a g e l l a
fo r m a tio n and in F i g s , 6 ,
7 , and 8 th e r e seem s t o be a g e n e r a l d is a m e a r a n o e o f g r a n u le s ,
w hereas i f th e g r a n u le s were a r e s u l t o f e y to n la s m ic a c t i v i t y ,
th e r e w ould be a la r g e r c o l l e c t i o n o f g r a n u le s in th e l a t e r
sta g e s o f f la g e lla
fo r m a tio n .
There i s no w e ll d e f in e d n u c le a r body t h a t c o u ld be
c o n n e c te d t o th e p o s s i b l e b l e o h a r o p l a s t , b u t t h i s d oes n o t
seem t o be a s e r i o u s o b j e c t io n in v ie w o f th e th e o r y t h a t th e
b a c t e r i a l n u c le u s i s d i f f u s e d th r o u g h o u t th e c e l l .
T h is
c o l l e c t i o n o f g r a n u le s c o u ld s t i l l c a r r y on n u c le a r a c t i v i t y
in r eg a r d t o f l a g e l l a fo r m a tio n .
A fte r th e n u c le a r fo r c e
t h a t moves th e g r a n u le s t o th e p o le s o f th e c e l l s , th e y need
n o t rem ain in c l o s e c o n t a c t w ith n u c le a r m a t e r ia l anymore to
e x e r t a f o r c e by w hich th e f l a g e l l a a r e form ed.
As soon as
th e g r a n u le s a re e n e r g iz e d , th e y may be a b le t o c a r r y on a lo n e
and may n o t n eed th e n u c le a r fo r c e f u r t h e r t o d e v e lo n f l a g e l l a
a t th e p o l e s .
The g r a n u le s in th e en d s o f th e c e l l seem t o be
th e c e n t e r o f e n erg y once th e y have c o l l e c t e d t h e r e .
I t must
be a d m itte d t h a t t h i s groun o f g r a n u le s or s i n g l e g r a n u le as
th e c a s e may be c o u ld be a tta c h e d to th e d i f f u s e d n u c le u s o f
th e c e l l , b u t in t h i s work no v i s i b l e a tta ch m en t h a s been
ob served .
In th e n e x t I l l u s t r a t i o n ( P i g . 2 ) one o f th e g r n u le a o f
th e group seem s t o be a c t u a l l y p u sh in g the plasm a membrane o f
27
th e c o l l o u t In f r o n t o f It*
I t i s l i k e l y t h a t t h i s o u term o st
g r a n u le o r th e plasm a membrane s h Ich I s pushed o u t ahead o f
I t la th e a c t u a l b e g in n in g o f th e f la g e llu m
I t s e l f , or Is
th e b a se from w hich th e f la g e llu m w i l l a r i s e .
There can a l s o
be n o t ic e d a g e n e r a l d i s t r i b u t i o n o f g r a n u la r m a t e r ia l th rou gh ­
o u t th e c e l l .
There I s l i t t l e
doubt a s to th e f u n c t io n o f th e
group o f g r a n u le s a t one end o f th e c e l l b eca u se o f th e ap­
p a r e n t c l o s e c o r r e l a t i o n b etw een th e g r a n u la r a c t i v i t y a t
th e end o f th e c e l l and th e f l a g e l l a
fo rm a tio n a t t h a t en d .
That f o r c e I s t h i s t h a t c a u s e s th e c o l l e c t i o n o f c e r t a i n
g r a n u le s a t th e p o le s o f th e c e l l ?
In some o f th e fu n g i th e
movement o f th e g r a n u le s and th e fo rm a tio n o f th e c i l i a
c l o s e l y accom p an ies th e n u c le a r or c e l l d i v i s i o n .
Prom th e c o n c lu s io n s rea ch ed In t h i s stu d y o f Sp, s e m e n s .
th e r e seem s t o be a n a tu r a l p r o g r e s s io n b e g in n in g w ith th e
r e p r o d u c tio n o f th e c e l l by d i v i s i o n and e n d in g w ith th e
c o m p lete fo r m a tio n o f th e f l a g e l l a .
The f l a g e l l a have been
o b se r v e d t o be form in g a t d i f f e r e n t s t a g e s o f c e l l d i v i s i o n .
I t I s b e l ie v e d t h a t a f t e r c e l l d i v i s i o n and when a f la g e llu m
h as s t a r t e d t o form , th e c e l l may b e g in to d i v i d e a g a in .
In t h i s I l l u s t r a t i o n ( P i g , 3 ) th e q u e s tio n a r i s e s a s t o
th e so u r c e o f th e p r o to p la sm ic th r e a d r e p r e s e n t in g th e new
f la g e llu m w hich p r o j e c t s outward from th e g r a n u le .
Is th is
a new ly form ed mass a r i s i n g from some v i t a l a c t io n o f th e
g r a n u le or I s i t a s h i f t or g a t h e r in g o f c y to p la s m ic m a te r ia l
28
from around th e granule*?
The w r i t e r b e l i e v e s t h a t g r a n u la r
a c t i v i t y c a u s e s a d e f i n i t e l y new x roto Iasm Ic mass t o be
form ed; t h a t t h i s e n e r g y used in g r a n u la r a c t i v i t y i s a c q u ir e d
from th e n u c le u s ; and th a t t h e s e g r a n u le s a c t a s s e p a r a t e ,
v i s i b l e p a r t s o f th e b a c t e r i a l n u c le u s .
As ca n be n o t ic e d t h i s c e l l ( P i g . 3) i s s m a lle r than
many o f th e o th e r s and may seem to be s l i g h t l y o u t o f p la c e
in th e g e n e r a l scheme b e c a u se in P i g . 2 and 4 th e r e are
s e v e r a l g r a n u le s w h ile in P ig . 3 th e r e la a p p a r e n tly o n ly o n e .
The sm a ll c e l l was p la c e d th e r e b e c a u se o f th e f i r s t a p p ea r an ce o f th e a c t u a l m ass o f m a te r ia l s t a r t i n g d evelop m en t
tow ard a f l a g e l lu m .
In P i g . 4 th e c e l l h a s form ed a d e f i n i t e b e g in n in g o f
th e f la g e llu m and h a s th e c h a r a c t e r i s t i c group o f g r a n u le s a t
its
b ase.
T h is i l l u s t r a t i o n a l s o shows th e f i r s t
I n sta n c e
where g r a n u le s may have e i t h e r moved o u t in t o th e f la g e llu m
or may have been c a r r ie d a lo n g w ith th e c y to p la sm accom panying
th e f u r t h e r d evelop m en t o f th e f l a g e l lu m .
As c a n be n o t i c e d ,
th e g r a n u le s are b e g in n in g t o s c a t t e r away from th e end o f th e
c e l l and seem t o be t h in n in g o u t .
In a l l p r o b a b i l i t y th e
f la g e llu m i s w e l l on i t s way t o b e in g formed and th e g r a n u le s
a r e m oving back i n t o th e body o f th e c e l l where th e y w i l l
p o s s i b l y be d i f f u s e d w ith th e o th e r n u c le a r m a t e r ia l o f th e
c e ll.
The c e l l in F i g . 5 i s shown to be In fu r th e r f l a g e l l a
29
fo r m a tio n w ith a more pronounced s c a t t e r i n g o f th e g r a n u le s
back from th e end and a l s o a g r e a t e r c o n c e n tr a t io n o f th e
g r a n u le s in th e f l a g e l l u m .
The f l a g e l l a a t th e o th e r end o f
th e c e l l h ave a lr e a d y form ed .
The f la g e llu m
.lu st form in g i s
shown t o be made up o f a s e r i e s o f g r a n u le s .-w h ic h a g r e e s
v e r y c l o s e l y w ith th e work o f L e if s o n ( 1 9 3 0 ) , a s he s t a t e d :
" I f o a r b o l f u c h s ln i s added t o a h a n g in g drop o f V ib r io s
(and o th e r organ ism s a s w e l l) some f l a g e l l a can he s e e n .
T hese f l a g e l l a are n o t s t a in e d s o l i d l y but a s a s e r i e s o f
d o t s ,"
T h is i l l u s t r a t i o n a l s o shows v e r y n i c e l y th e way in w hich
th e c e l l d i v i d e s .
B e fo re c o n s t r i c t i o n ta k e s p l a c e , a p l a t e
or a r in g o f d e n s e r p r o to p la sm ic m a t e r ia l i s form ed a t th e
p o in t o f d i v i s i o n .
T h is phenomenon was o b serv ed many t im e s .
F i g s . 6 and 7 may be ta k en t o g e t h e r b e c a u se th e y show
th e same s t a g e o f d e v e lo p m en t.
A gain t h e s e c e l l s a r e youn ger
and have n o t d e v e lo p e d a lo n g w ith t h e i r f l a g e l l a .
The f la g e llu m
o f e a ch o f t h e s e c e l l s seem s t o h a v e a mass o f c y to p la s m ic
m a t e r ia l a t each t i p , t h ic k e r and d e n s e r than th e r e s t o f th e
f l a g e l lu m .
T h is t i p d id n o t ap p ear t o be a s d en se a s th e
g r a n u le s In th e c e l l s th e m se lv e s and t h e r e f o r e i s th ou gh t t o
c o n s i s t n o t Cfa n u c le a r g r a n u le b u t o f a d e n ser mass o f c y to ­
p la sm .
T h is c o u ld be compared v e r y e a s i l y and c l o s e l y to th e
work on th e c i l i a
fo r m a tio n o f some o f th e f u n g i ,
h ow ever, i t
w ould be hard t o sa y w h eth er o r n o t t h i s mass a t th e t i p i s
30
c a u se d by th e w h ip p in g a c t i o n o f th e f la g e llu m .
T h is would
seem p r o b a b le i f th e f l a g e l l a form o u l t e f a s t .
In P ig . 8 th e u p per f la g e llu m Ia f u l l y d e v e lo p e d as a
s i n g l e f l a g e l lu m .
a can be n o t ic e d th e r e la s t i l l a g r a n u le
rem a in in g a t th e b a se o f the f la g e llu m w hich h a s moved out
in to i t s l i g h t I y .
The f l a g e l l a
o f Sp. serp ona form a s a s o l i d
mass and th e n s p l i t i n t o many s m a lle r t h r e a d - l ik e f l a g e l l a
— P ig . BA.
I t seem s t h a t th e g r a n u le s out In th e f l a g e l l a
have some v i t a l c o n n e c tio n w ith t h i s s p l i t t i n g p r o c e s s .
No
c o n c lu s iv e e v id e n c e h a s b een found t o s t a t e th a t th e g r a n u le s
move down th e f la g e llu m a s i t s p l i t s
in t o many s m a lle r s t r a n d s .
L e lfa o n (1 9 3 0 ) and Puhrmann (1 9 1 0 ) have a l s o d em on strated
g r a n u le s in th e f l a g e l l a o f b a c t e r i a .
The g r a n u le s e i t h e r
r e c e d e d u rin g th e s p l i t t i n g p r o c e s s or th e y b reak up in to
s m a lle r p a r ts c o n ta in e d In each th r e a d as th e one f la g e llu m
s p lits
i n t o many f l a g e l l a .
In P i g . 12 th e f l a g e l l a have
a lr e a d y s p l i t but s t i l l c o n ta in a la r g e p r o p o r tio n o f g r a n u la r
m a t e r i a l.
E it h e r th e g r n u le h a s n o t moved b a ck t o th e c e l l
b u t w i l l in th e fu tu r e or i t may be composed o f s m a lle r
g r a n u le s w hich may s e p a r a te as th e f la g e llu m s I l t s
s m a lle r u n i t s .
In to
P i g . 8 i s rem arkable b eca u se th e c e l l was
s t a i n e d J u st uw th e moment t h a t th e f la g e llu m a t one p o le had
j u s t d e v e lo p e d t o th e s ta g e b e fo r e s p l i t t i n g and a t th e o th e r
p o le th e f la g e llu m was J u st b e g in n in g t o s p l i t .
Ae can be
n o t i c e d , th e f l a g e l l a b e g in t o s p l i t a t th e d i s t a l end and
31
s p i l t down t o th e c e l l ,
In F l g s e 6 , 1 0 , 1 1 , and 15 th e r e onn be n o t ic e d a
phenomenon In w hich th e g r a n u le moves o u t away from th e c e l l
and rem ains a t th e b a s e o f th e f l a g e l lu m , form ing th e apex o f
a cone.
T h is c o u ld p o s s i b l y bo c a u se d by th e g r a n u le moving
away and p u l l i n g o u t o f p la c e a membrane from th e c e l l .
T h is
con e c o u ld a l s o be c a u se d by a d e f i n i t e phase o f grow th w here­
a s a t h ic k e r p a r t o f th e f la g e llu m was ca u sed t o form b etw een
th e g r a n u le and th e c e l l w a l l .
T h is g r a n u le a t th e t i p o f
th e c o n e -sh a p e d mass I s In ap- e a ra n ce v e r y much l i k e the
b I e pharo p l a s t o f th e fu n g i and o th e r m o t ile p la n t and anim al
c e lls .
Could i t be t h a t th e w hole b a c t e r i a l c e l l
la th e
n u c le u s and t h a t t h i s h ig h ly r e f r a c t i v e body ( b le n h n r o n la s t)
has moved away from I t ?
N o tic e th a t t h i s phenomenon ta k e s
p la c e a p p a r e n tly In o n ly th e o ld e r c e l l s .
In th e upper p o r t io n
o f F i g . 11 can be s e e n what s e e m in g ly I s a f la g e llu m a r i s i n g
from th e c o l l by two d i f f e r e n t s tr a n d s and th e n u n i t i n g .
: l g . 13 I s e x tr e m e ly i n t e r e s t i n g as I t she,.,a a d i f f e r e n t
phenomenon w h ich t o th e w r i t e r ' s know ledge h as n e v e r been
o b se r v e d b e f o r e .
In some o f th e c u l t u r e s a p p r o x im a te ly tw e n ty -
fo u r h o u rs o ld th e r e c o u ld be s e e n s m a ll c o e c u s - l l k e m asses
w ith a group v.L f l a g e l l a
com ing o u t from th e c e n t r a l p a r t i c l e .
A f t e r s e v e r a l s l i d e s o f s t a in e d s u s p e n s io n s o f organ ism s were
made i t was c o n c lu d e d t h a t t h e s e sm a ll forms w ere t i p s broken
o f f from c e l l s w ith th e f l a g e l l a s t i l l a t t a c h e d .
N a tu r a lly
32
th e n e x t move was t o t r y t o f in d a s t a g e in w hich th e t i n was
J u st b r e a k in g o f f and In t h a t way c o n fir m th e t h e o r y .
F ig , 13
a p p a r e n tly r e p r e s e n t s a c e l l t h a t I s J u st " b low in g I t s c a p s" .
Tlie la r g e dark p la c e s a t b oth en d s th a t seem a b o u t t o break
o f f a re n o t v e r y much d ark er th an th e r e s t o f th e c e l l and
a r e n o t n e a r ly as dark a s th e g r a n u le s found in c o n n e c tio n
w ith f l a g e l l a fo r m a tio n .
A p p a ren tly th e c e l l h a s reach ed a
c e r t a i n s t a g e o f normal or abnorm al g ro w th , p erh ap s cau sed
by e n v iro n m en ta l f a c t o r s , t h a t have b rou gh t on t h i s phenomenon
o f lo s in g i t s c e l l t ip s .
F i g s , I t o 8A I n c l u s i v e a r e d raw ings o f d i f f e r e n t b a c t e r ia
from s i x , s i x and a h a l f , and se v en hour c u lt u r e s and show
t h a t th e f l a g e l l a a r e d e v e lo p in g most r a p id ly a t t h a t a g e ,
w h ile ^ l g s . 6 t o 13 I n c l u s i v e a r e draw ings made o f d i f f e r e n t
b a c t e r ia from a p p r o x im a te ly tw e n ty hou r c u l t u r e s .
The you n ger
c e l l s s t a i n l i g h t e r th a n th e o ld e r on es and t h e r e f o r e are
r e p r e s e n te d by l i g h t e r d r a w in g s.
From th e o b s e r v a tio n s and e v id e n c e accu m u lated In t h i s
i n v e s t i g a t i o n th e seq u en ce o f e v e n ts t h a t ta k e s p la c e In th e
fo r m a tio n o f f l a g e l l a
I.
in 3 p , se r p e n s ap p ears t o b e as f o llo w s :
An a c c u m u la tio n o f g r a n u le s o c c u r s in one end o f th e c e l l
p r e lim in a r y to f l a g e l l a fo r m a tio n a t t h a t e n d .
T h is s tim u lu s
t h a t s t a r t s th e p o la r a c cu m u la tio n o e c u r ln g som etim e a f t e r
c e l l d i v i s i o n la th o u g h t to a r i s e from n u c le a r a c t i v i t y .
A lthough t h e r e h as n o t been o b ser v ed any a p p aren t c o n n e c tio n
b etw een th e d i f f u s e d n u c le u s o f th e c e l l and th e g r a n u le s ,
t h e s e g r a n u le s may a c t u a l l y be p a r t s o f th e ch rom atin m a te r ia l
o f th e b a c t e r i a l c e l l ,
2.
The f i r a t str a n d o f th e f la g e llu m a r i s e s d i r e c t l y c u t from
one o r more o f th e p o la r g r a n u le s and p o in t s away from th e
c e ll.
T h is str a n d becom es lo n g e r and t h i c k e r .
G ran u les have
b een o b ser v ed l y i n g o u t In th e f la g e llu m soon a f t e r i t a p p e a r s.
3.
-Ih lle th e s i n g l e
f la g e llu m la d e v e lo p in g th e r e o ccu rs a t
its
t i p a s l i g h t l y l a r g e r mass o f proto? Iaein,
T h la knob a t
th e t i p I s a p p a r e n tly n o t as d en se as th e g r a n u le s p r e v io u s ly
d e s c r ib e d b u t seem s t o be a c o n c e n tr a t io n o f th e f l a g e l l a r
c y to p la s m ,
4.
A f t e r th e s i n g l e f la g e llu m h as rea c h e d a c e r t a i n s ta g e o f
grow th whereby i t la a p p r o x im a te ly h a l f as lo n g as th e c e l l ,
i t b e g in s t o s p l i t I n to many th in n e r s t r a n d s .
From th e
w r i t e r ' s o b s e r v a tio n th e s i n g l e f la g e llu m s p l i t s
from th e
d i s t a l end down t o th e p o in t o f a tta ch m en t u n t i l I t becomes
a f u l l y d e v e lo p e d t u f t o f f l a g e l l a .
F u r th e r s t u d i e s In t h i s f i e l d may o f c o u r se le a d to some
r e v i s i o n o f th e s ta te m e n ts a b o v e .
34
StWARY AND CONCLUSIONS
T h is s tu d y In a d d it io n t o d e v e lo p in g a new s t a i n i n g
t e c h n ic In th e d i f f e r e n t i a t i o n o f th e s tr u c t u r e o f th e f l a g e l l a
o f b a c t e r ia h a s d e f i n i t e l y sh o rn a number o f s t a g e s In th e
c y t o l o g l c a l d evelop m en t o f th e f l a g e l l a o f Sn. s e m e n s .
S e r o l o g i c a l p r o o f o f th e a n t i g e n i c d if f e r e n c e b etw een th e
f l a g e l l a and th e soma i s p r e s e n t e d .
T h is d i f f e r e n c e I s e x ­
p la in e d on th e b a s i s o f th e d i f f e r e n c e In p r o p o r tio n o f
s u r f a c e t o c y to p la s m ic c o n te n t w hich e x i s t s in th e f l a g e l l a
and th e main or "eoma" p a r t o f th e c e l l .
1.
A t e c h n ic h a s b een d e v e lo p e d by w hich p o la r g r a n u le s
in th e c e l l s o f S o . se r p e n s may be s t a i n e d ,
2.
The g r a n u le s In th e ends o f th e c e l l s w ere found, to
accompany f l a g e l l a fo r m a tio n .
v.
G ran ules l y i n g in th e f l a g e l l a were a l s o s t a i n e d .
4.
The f l a g e l l a
a r is e c l o s e l y a l l i e d w ith g r a n u la r
a c t i v i t y a s new s t r u c t u r e s w ith d i f f e r e n t s e r o l o g i c a l p r o p e r t ie s
from th e p r o to p la sm o f th e c e l l ,
5.
The f l a g e l l a have been found t o be form in g a t a l l
d i f f e r e n t s t a g e s o f c e l l d i v i s i o n and d e v elo p m en t,
6.
The f l a g e l l a a r i s e a s s i n g l e s t a l k s and th en s n l i t
I n to many t h r e a d - l i k e f l a g e l l a .
From th e w r i t e r ’ s o b se r v a ­
t i o n s th e y u s u a l l y s p l i t from th e d i s t a l end down t o th e p o in t
o f a tta c h m e n t.
7.
S e r o lo g i c a l s t u d i e s
( p r e c i p i t i n r e a c t i o n ) were made
SB
o f Sil«_ ocrpeno u s in g th r e e d i v i s i o n s o f th e c e l l — th e f l a g e l l a ,
th e Bom and th e w hole c e l l s ,
8.
S e r o l o g i c a l t e s t s show n d i f f e r e n c e in th e a n t ig e n ic
s t r u c t u r e b etw een th e soma and f l a g e l l a .
9,
Tht f l a g e l l a
c o n ta in a much g r e a t e r amount o f
p r e c i p i t i n o g e n lc m a te r ia l th an th e soma,
10.
Tho s or a c o n ta in a su b s ta n c e c a p a b le o f r e a c t in g
VfI h th e f l a g e l l a r - p r e c i p i t i n in th e Imcrune serum .
A.cknm-IedgBiOnt. -..The w r it e r w ish e s t o e x p r e s s h i s a p p r e c ia t io n
f o r th e many h e l p f u l s u g g e s t io n s made by Dr. F , B . C otner who
s u g g e s t e d t h i s problem ; f o r th e a d v ic e and in v a lu a b le c r i t i c i s m
g iv e n by Dr. D, B , S w in g le ; and f o r th e keen I n t e r e s t shown
by D r. B. T,. Johnson and F r , Je He P epper d u rin g th e p r o g r e ss
o f t h i s work.
36
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38
E x p la n a tio n o f P l a t e I
H ieae d raw in gs from s t a in e d s i Id ea o f Sda aarpena were
made t o th e same s c a l e w ith In d ia Ink under a la r g e r e a d in g
g l a s s and r e p r e s e n t a m a g n if ic a t io n o f 6 ,3 6 0 tim e s th e o r i g i n a l
s i z e o f th e c e l l s a s m easured by an o c u la r m ic r o m e te r .
In F i g s ,
I t o 7 i n c l u s i v e a 2 mm. Z e is s ap ooh rom atle 1 .4 0 n u m erica l
a p e r tu r e o b j e c t i v e , a p a ir o f IOx Z e is s ap ooh rom atle o c u la r s ,
ar I a 1 ,4 0 n u m e r ic a l a p e r tu r e c o n d e n se r were u s e d ,
E lg , I ,
C e ll from a 6 1 /2 h ou r c u lt u r e w ith a group o f
g r a n u le s a t one end p r e lim in a r y t o th e ap p earan ce o f th e
f l a g e l lu m .
F lg e 2 ,
A c tu a l s i z e 11 x 1 ,3 m ic r o n s .
F ere a group o f g r a n u le s a t one end o f th e c e l l
h a s c o l l e c t e d , o f w hich one g r a n u le I s a c t u a l l y p r o tr u d in g
o u t p a s t th e n a t u r a l boundary o f th e end o f th e c e l l and seem s
t o be a c t u a l l y p u sh in g th e plasm a membrane o f t h i s p a r t o f
th e c e l l o u t In f r o n t o f I t ,
L ess d e n se g r a n u le s a r e s c a t ­
t e r e d th ro u g h o u t th e c e l l which Is from a 6 1 /2 h ou r c u l t u r e .
A c tu a l s i z e 11 x 1 ,1 m ic r o n s,
P ig . 3 ,
A s i n g l e g r a n u le a p p ea r s a t th e end o f th e c e l l
from w h ich a sm a ll str a n d o f p r o to p la sm Is p r o tr u d in g .
I s th e f i r s t ap p earan ce o f th e f l a g e l lu m ,
At th e o th e r end
a re two sm a ll g r a n u le s , p o s s i b l y th e f i r s t o f a grou
g a th e r ,
Tlie c e l l I s from a 7 hour c u l t u r e .
T h is
to
A c tu a l s i z e
6 ,5 x 1 ,0 m ic r o n s ,
F ig . 4 ,
T ills I s th e f i r s t c e l l t o a c t u a l l y have a g r a n u le
39
o u t In th e f l a g e l lu m .
The str a n d o f f la g e llu m I s la r g e r than
In P ig , 3 and th e r e a re a few g r a n u le s s c a t t e r e d th rou gh ou t
th e c e l l .
S ta in e d from a 7 hour c u l t u r e .
A c tu a l s i z e 11 x 1 .1
m ic r o n s .
P ig , 5 ,
A la r g e group o f g r a n u le s appear a t th e end o f
th e c e l l and many in th e f la g e llu m ,
end o f th e c e l l h ave a lr e a d y form ed .
^he H t g e l l a a t th e o th e r
The c e l l I s a p p a r e n tly
in th e f i r s t s t a g e o f d i v i s i o n w ith a p l a t e or r in g form ing
n e a r th e m id d le .
S ta in e d from a 6 1 /2 hour c u l t u r e .
A c tu a l
s i z e 9 ,5 x 1 ,1 m ic r o n s.
P ig . 6 .
A c e l l w ith a l o n g , s l e n d e r , s i n g l e f la g e llu m
h a v in g a s l i g h t l y la r g e r mass o f p ro to p la sm a t th e t i p ,
At
th e o th e r end th e r e i s a sm a ll c o l l e c t i o n o f g r a n u le s .
Prom
a 7 hour c u l t u r e .
F"lg* 7 .
P ig , 6 ,
A c tu a l s i z e 5 ,5 x 0 .9 m ic r o n s.
\ c e l l w ith a f la g e llu m v e ry s i m i l a r t o th a t o f
S ta in e d from a 7 hour c u l t u r e .
A c tu a l s i z e o f th e
c e l l in P ig , 7 i s 8 x 0 ,9 m ic r o n s .
8,
The f l a g e l l a o f t h i s c e l l are in two d i s t i n c t
s t a g e s o f d e v e lo p m e n t.
In P ig , 8 th e f la g e llu m i s s t i l l
s i n g l e w ith a g r a n u le in I t s b a s e .
In P ig . 8A th e s i n g l e
f l a g e l l u m , w hich h a s a g r a n u le a t i t s b a s e , h a s s t a r t e d t o
s p l i t I n to s e v e r a l t h in n e r f l a g e l l a .
c u ltu r e .
S ta in e d from a 6 hour
A c tu a l s i z e 11 x 1 .3 m ic r o n s .
P ig , 9 .
The f la g e llu m h as a s i n g l e g r a n u le a t i t s b a s e .
T h is g r a n u le i s ou t from th e c e l l and a t th e apex o f a cone
40
c o n n e c tin g i t t o th e c e l l .
Thia la v e r y s i m i la r t o th e
b le p h a r o p la s t o f c e r t a i n f u n g i.
la te d .
T h is c e l l i s h i g h ly v a cu o ­
S ta in e d from an 18 hou r c u l t u r e .
A c tu a l s i z e 5 .3 x 1 .2
m ic r o n s .
P ig . 1 0 .
t h a t o f P ig . 9 .
The upperm ost f la g e llu m i s v e r y s i m i l a r to
T h is c e l l i s a l s o h ig h ly v a c u o la te d and h a s
s e v e r a l g r a n u le s s c a t t e r e d th r o u g h o u t.
hour c u l t u r e .
P ig . 1 1 .
S ta in e d from an 18
A c tu a l s i z e 8 .3 x 1 .2 m ic r o n s.
At th e up ©r end o f t h i s c e l l th e f la g e llu m
i s p e c u l i a r l y form ed .
The f la g e llu m seems to come from th e
c e l l in two s e p a r a te s tr a n d s w hich a r e soon j o in e d .
The
f la g e llu m a t th e lo w e r end i s s i m i la r t o th o s e i n ^ lg a , 9
and 1 0 .
The c e l l I s a p p a r e n tly n o t v a c u o la te d b u t h as s e v e r a l
g r a n u le s s c a t t e r e d th ro u g h o u t th e c e l l .
hour c u l t u r e .
Pig* 1 2 ,
th r o u g h o u t.
S ta in e d from an 18
A c tu a l s i z e 7 ,5 x 1 .0 m icr o n s.
H ig h ly v a c u o la te d c e l l w ith many g r a n u le s
There a r e a l s o many g r a n u le s in th e f l a g e l l a .
S ta in e d from a 22 hour c u l t u r e .
P ig . 1 3 .
A c tu a l s i z e 1 0 ,5 x 1 .3 m ic r o n s .
The f la g e llu m o f th e upper end h a s a g r a n u le
s i m i la r t o th o s e in P i g s . 9 , 1 0 , and 11 which form s th e apex
o f a cone.
In F ig . 13 th e t i p s o f th e c e l l seem about to
b rea k o f f .
S ta in e d from an 18 hou r c u l t u r e .
1 0 .5 x 1 ,3 m ic r o n s .
A c tu a l s i z e
41
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