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STUDIES ON METABOLISM OF TAENIA TAENIAEFORMIS (BATSCH, 1786) WOLFFHUEGEL, 1911

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ZD9 5 5
V7il.no t h , Ja. j e s Her a.-nan, 1 9 1 0 S t u d i e s on 'nctabolis-n o f Taenia
t a e n i a e f o r . a i s ( S a t s c h , 1 7 8 6 ) ’7 o l f f h u e , r ; e l ,
1 9 1 1 ...
hew Y o r k , 1 9 4 2 .
84 t y p e v / r i t t e n l e a v e s .
29cn.
tab les, d i ^ r s .
T h o s l 3 ( ? h . S . ) - l e w Y ork u n i v e r s i t y ,
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5:tbl i o / ; r a p h y ; p . 7,3 - 8 4 .
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RECEIVED.
STUDIES ON METABOLISM OF TAENIA TAENIAEFORMIS
(BATSCH,1786) W o lffh u eg el, 1911
James H. Wilmoth
Departm ent o f B iology
U n i v e r s i t y C o lle g e , New York U n i v e r s i t y
S u b m itted i n p a r t i a l f u l f i l l m e n t o f t h e re q u ire m e n ts f o r
t h e d e g re e o f D octor o f P h ilo so p h y a t New York U n i v e r s i t y .
J u n e , 1942
INTRODUCTION
The b io lo g y o f th o s e i n t e r n a l p a r a s i t e s w ith
f i n e l y a d ju s te d h o s t s p e c i f i c i t y I n v i t e s a t t e n t i o n t o phys­
i o l o g i c a l problem s which a r e c l o s e l y i n t e g r a t e d w ith th o s e
o f t h e r e s p e c t i v e h o s t and y e t which a r e p e c u l i a r l y u n iq u e .
When a tte m p ts a r e made to c u l t u r e p a r a s i t e s i n v i t r o , t h e s e
r e l a t i o n s h i p s p ro v id e numerous c lu e s on th e o ne h an d , and
a t t h e same tim e i n c r e a s e the co m p lex ity to t h e p o i n t o f b a f­
fle m e n t on t h e o t h e r .
The te c h n iq u e s o f t i s s u e c u l t u r e have
overcome some o f th e b ro a d e r p ro blem s o f in v i t r o en v iro n m en t,
and t h e s e f a c t s have been and p ro b a b ly w i l l t o an i n c r e a s ­
in g e x t e n t be a p p lie d to t h e i n v i t r o c u l t u r e o f t i s s u e
p a r a s i t e s , and i n a l e s s e r d eg ree t o o t h e r i n t e r n a l p a r a s i t e s .
The h i s t o r y of t i s s u e c u l t u r e marks i t s g e n e s is
w ith t h e famous ex p e rim e n ts o f Ross H arriso n (1907, 1910)
i n which s u c c e s s f u l c u l t i v a t i o n and growth o f frog n erv o u s
t i s s u e immersed in a hanging drop o f lymph was d e m o n stra te d .
Sm all fragm en ts o f t i s s u e from th e c e n t r a l nervous system
were suspended i n lymph from c o v e r - s l i p s over hollow -ground
slid e s.
T h is i s t h e fundam ental " h a n g in g -d ro p method" which
has see n wide u s a g e .
T h is method a s employed by Lewis and
Lewis (1911 and 1912) i s p e rh a p s t h e s im p le s t te c h n iq u e f o r
W & 'S I 6
-2 -
f o r o b se rv in g t i s s u e a c t i v i t y i s o l a t e d from t h e o rg an ism .
The Lewis* employed a s p e c i a l p h y s i o l o g i c a l s a l i n e s o l u ­
t i o n w ith or w ith o u t t h e a d d i t i o n o f c h ic k e n b o u i l l o n .
Such a method a llo w s f o r g r e a t e r u n if o r m ity by e l i m in a ­
t i o n o f many o f t h e unknowns e x c e p t th o s e r e s u l t i n g from
t h e a c t i v i t y o f th e t i s s u e i t s e l f .
^
C a r r e l and Burrows m o d ifie d t h i s p ro c e d u re by
r e g u l a r and f r e q u e n t s u b - c u l t u r i n g i n which p a r t o f t h e
e x p la n te d t i s s u e i s removed and p la c e d i n f r e s h medium.
Under su ch c o n d i t i o n s , m u l t i p l i c a t i o n i s v e ry r a p i d . The
C a r r e l f l a s k method p e r m its t i s s u e s t o grow i n media f o r
lo n g e r p e r io d s o f tim e th a n i s th e c a s e i n th e p r e v io u s ly
m entioned t e c h n iq u e s .
Out)
In t h e te c h n iq u e s p e r f e c t e d by Maximow, th e
c o v e r - s l i p s w ith a t t a c h e d t i s s u e s a r e n o t d i s t u r b e d , but
t h e e x p la n ts a r e washed i n s a l i n e i n s i t u and s u p p lie d
w ith f r e s h medium a t r e g u l a r i n t e r v a l s .
In c o n tra s t to
A
th e C a r r e l and Burrows method, c o m p a ra tiv e ly l i t t l e c e l l u ­
l a r m u l t i p l i c a t i o n o c c u rs ,a n d t h i s f e a t u r e i s u t i l i z e d w ith
t h e C a r r e l f l a s k to keep t i s s u e s f o r s e v e r a l months w ith
i n f r e q u e n t washings and medium chan ges.
V ario u s a tte m p ts have been made to p ro v id e f o r
c o n s ta n t movement and change o f th e medium.
These can be
c l a s s i f i e d i n a group a s the p e r f u s i o n and r o l l e r - t u b e
m ethods.
Bloom (1937) made a d e e p - s e a te d c r i t i c i s m when
-3 -
he s t a t e d t h a t na l l o f t h e s e t e c h n i q u e s , w ith t h e p o s s i b l e
e x c e p tio n o f some o f t h e p e r f u s i o n m ethods, s u b j e c t t h e ex­
p l a n t e d c e l l s t o a v ery abnormal environm ent i n which t h e
accum ulated m e t a b o l i t e s a r e o n ly removed a t i n t e r v a l s o f
s e v e r a l days and n o t c o n tin u o u s ly as by th e c i r c u l a t i n g
blood and lymph w ith in th e body".
I t i s in te r e s tin g to
n o te t h a t Wardle (1937a) made a s ta te m e n t o f s i m i l a r s i g ­
n i f i c a n c e r e g a r d in g d a t a on p h y s i o l o g i c a l p r o c e s s e s o f
h e lm in th s which had been g a th e r e d l a r g e l y from i n v i t r o
stu d ie s.
The method o f t i s s u e c u l t u r e has been a p p l i e d t o
a v a r i e t y o f problem s u s i n g b o th em bryonic and a d u l t t i s ­
sues o f v e r t e b r a t e s .
I n v e r t e b r a t e and p l a n t t i s s u e s have
been c u l t i v a t e d t o a l e s s e r e x t e n t ,
l f u e l l e r (1929) u t i l ­
iz e d t h e h a n g in g -d ro p method f o r c u l t u r i n g l a t e r a l l i n e
o rg an t i s s u e o f A s c a r i s i n a s tu d y o f f a t m etab o lism . Chung
(1936) a tte m p te d t o grow S tr o n g y lo id e s l a r v a e i n t i s s u e
c u ltu re s.
And somewhat c o n v e r s e ly , Rix and Laas (1936)
s t im u la t e d grow th o f c h ic k h e a r t and mouse lu n g w ith f r e s h
A s c a r is and T ae n ia s a g l n a t a e x t r a c t s .
I n r e c e n t y e a rs s e v e r a l rev iew s upon t h e s u b j e c t
o f h e lm in th p h y s io lo g y have a p p e a re d , (von B rand, 1934;
McCoy, 1935; W ardle, 1 9 3 7 ).
A ls o , re v ie w s o f a more gen­
e r a l n a t u r e , n e v e r t h e l e s s , h a v e c o n s i d e r a b l e m a t e r i a l on
th e p h y s io lo g y o f p a r a s i t e s , ( P e t e r s , 1936; H o e p p li,1 9 3 7 ;
-4 -
and H o e p p li, Feng, and Chu, 1 9 3 8 ).
I n v i t r o c u l t u r e , b ein g
what i t i s , has caused f i r s t em phasis t o be throw n upon t h e
re q u ir e m e n ts o f m etab o lism , e s p e c i a l l y r e s p i r a t i o n and nu­
tritio n .
T here i s s t i l l c o n s i d e r a b l e c o n tr o v e r s y co n c ern ­
in g w hether th e r e s p i r a t i o n o f i n t e r n a l p a r a s i t e s i s a e r o b ic
o r a n a e r o b ic .
The environm ent o f i n t e s t i n a l p a r a s i t e s i s
e x c e e d in g ly low i n oxygen.
Von Brand (1938) h a s com piled
t h e r e s u l t s o f v a r io u s i n v e s t i g a t o r s on t h i s s u b j e c t .
P a r a s i t e s l i v i n g i n t h e i n t e s t i n e would have t o u t i l i z e
oxygen a t a v ery low t e n s i o n , o r p o s s e s s an a n a e ro b ic ty p e
o f m etabolism o r b o th .
I n 1883, Bunge made th e f i r s t im­
p o r t a n t i n v e s t i g a t i o n s o f a n a e r o b l o s i s i n which he was a b le
t o keep th e a s c a r i d s o f t h e c a t a l i v e fo u r t o s i x days i n
t h e ab se n ce o f oxygen.
B eginning w ith t h e work o f Weinland (1901, 190li),
a t t e m p t s were made to i n d i c a t e t h a t i n t e s t i n a l en to z o a
p o s s e s s e d an a n a e ro b ic m etab o lism .
Weinland b e lie v e d g ly ­
cogen t o be th e p r i n c i p a l m a t e r i a l broken down, ( a t l e a s t
i n t h e c a s e o f A s c a r is ) s i n c e i t c o n s t i t u t e s about 33 p e r
c e n t o f the d r y weight o f t h e worms.
Matthews (1936)
s t a t e d , " th e d e x t r o s e , which i n an a n a e ro b ic medium th u s
f u n c t i o n s i n t h e p l a c e o f oxygen as an o x i d i z i n g a g e n t,
g iv e s r i s e t o a lc o h o l when i t th u s f u n c t i o n s , or t o f a t t y
a c i d s , i n p l a c e o f an a ld e h y d e ."
Weinland found o n ly v a l -
-5 -
e r i c a c i d , b u t F l u r y (1 9 1 2 ); F i s c h e r (1 9 2 4 ); and von
Brand (1954) have shown t h a t v a r io u s low f a t t y a c i d s ,
a s w e ll as a sm a ll amount o f l a c t i c a c id a r e formed by
t h e a n a e r o b ic f e r m e n ta tio n o f g lu c o se i n A s c a r i s . Toryu
(1936) w ith t h e h o rs e A s c a r is (P a r a s c a r i s eauorum * s v n . .
A s c a r is m eealo cen hala) found an a n a e ro b ic m etabolism sim i­
l a r to t h a t von Brand and o t h e r s found w ith £ . l u m b r i c o l d e s .
I n 1926, tfein lan d and von Brand s tu d ie d th e an­
a e r o b i c m etabolism o f t h e sheep l i v e r f l u k e , F a s c i o l a
h e n a t l e a , and came t o th e c o n c lu s io n t h a t i t g ained en erg y
u n d er a f e r m e n ta tio n s i m i l a r t o t h a t o f A s c a r i s , but t h a t
t h e f a t t y a c id s produced were o f a h i g h e r , n o n - v o l a t i l e
ty p e.
H a rn isc h (1932) added c o n firm a to ry ev id e n c e f o r th e
work o f Weinland and von Brand and d em o n strated t h a t th e
amount o f carbon d io x id e produced by F a s c i o l a h e n a t i c a i s
th e same u n d er a e r o b i c o r a n a e ro b ic c o n d i t i o n s .
Von Brand (1929, 1935) found t h a t he could keep
t h e tapeworm, M oniezla exnansa
a l i v e f o r a number o f hours
when removed from i t s normal h a b i t a t .
He made com p arativ e
a n a ly s e s o f worms j u s t removed from t h e sheep i n t e s t i n e w ith
th o s e k e p t i n R in g er s o l u t i o n f o r s i x h o u rs .
He found a
d e c r e a s e of g ly co g en c o n te n t amounting t o o n e - f o u r t h o f one
p e r c e n t un d er t h e s e c o n d i t i o n s , w h ile f a t c o n te n t rem ained
c o n s t a n t . In 1931, A lt and T i s c h e r s tu d ie d t h e same p a r a s i t e
and i n v i r t u a l agreem ent w ith von B rand, showed t h a t f a t t y
a c id s and l a c t i c a c id i n alm ost e q u a l amount make up most o f
-6 -
t h e f e r m e n ta tiv e p ro d u c ts o f t h i s worm.
H a rn isc h (193»)
s t u d ie d T ria e n o o h o ru s n o d u lo s u s . a c e s to d e from a f r e s h ­
w ater f i s h and found t h a t oxygen a b s o r p tio n was p r o p o r t i o n ­
a l to i t s p a r t i a l p r e s s u r e i n th e s u rro u n d in g medium; b u t
t h a t carb o n d io x id e e x c r e te d bore no r e l a t i o n s h i p t o th e
amount o f oxygen a b s o rb e d .
He su g g e ste d t h e h y p o th e s is
t h a t t h e oxygen consum ption o b serv ed may r e p r e s e n t " th e
f u l f i l l m e n t o f an oxygen d e b t a r i s i n g from t h e a c cu m u la tio n
o f o x i d i z a b l e s u b s ta n c e s produced d u rin g a n a e ro b ic m etab o l­
ism and n o t c o m p le te ly removed from t h e body".
T h is i n d i ­
c a t e s a r e l a t i o n s h i p s i m i l a r t o t h a t s tu d ie d by L e s s e r i n
t h e earthworm and b r i e f l y d e s c r ib e d by Rogers (1 9 2 7 ).
L e s s e r found t h a t t h e earthworm produced f a t t y a c id s and
carbon d io x id e i n t h e absence o f oxygen.
When a i r i s ad­
m itte d t o th e earthworms a f t e r such an a n o x y b io tic p e r i o d ,
he o b serv ed a v e ry h ig h oxygen i n t a k e w ith a v e ry low
re s p ir a to ry q u o tie n t.
D e s p ite t h e s e a u t h e n t i c I n v e s t i g a t i o n s , t h e r e
a r e a number o f workers who q u e s tio n w hether A s c a ris i s
an a n a e ro b ic an im a l.
S l a t e r (1925) k e p t A s c a r is i n oxygen-
f r e e s a l t s o l u t i o n s a t 37° C.
He found t h a t t h e worms
became i n a c t i v e , b u t rem ained a l i v e f o r a s lo n g a s s i x d a y s .
Upon exposure t o a i r , a c t i v i t y was resum ed.
With o t h e r
a s c a r i d s u n d e r s i m i l a r a n a e ro b ic c o n d i t i o n s , but p e r i o d i ­
c a l l y s t i m u l a t e d to movement by m ild i n d u c t i o n sho cks, th e
-7 -
e x te n t o f l i f e was 24 t o 48 h o u r s .
C o n tro ls s i m i l a r l y
s t im u la t e d b u t exposed t o a i r c o n tin u e d to l i v e . S l a t e r
came to th e c o n c lu s io n t h a t A s c a r i s , when d e p r iv e d o f
oxygen, can re d u c e i t s m e ta b o lic r a t e t o a v e ry low v a lu e
and go i n t o a s t a t e o f "suspended a n im a tio n " .
M u eller (1929) found t h a t f a t s to r e d i n t h e l a t ­
e r a l l i n e t i s s u e of A s c a r is d is a p p e a re d when p i e c e s o f t h i s
t i s s u e were c u l t u r e d by t h e h an g in g -d ro p method and he con­
c lu d e s t h a t normal f a t m etabolism o c c u r s .
He b e l i e v e s t h a t
t h i s i n d i c a t e s an a e r o b i c m etabolism f o r A s c a r is and i n
c o n t r a s t t o th e t h e o r y o f t h e W einland-von Brand sc h o o l
who co n clu d e t h a t f a t s a r e e x c r e t o r y p r o d u c ts .
Adam (1932) d e m o n stra te d t h a t A s c a r i s u t i l i z e d
oxygen from the s u rro u n d in g medium; and Harwood and Brown
(1933) showed t h a t , u n d e r s ta n d a r d c o n d i t io n s o f te m p e r a tu r e
and p r e s s u r e , fem ale A s c a ris u t i l i z e d 0 .0 3 t o 0.045 c c . o f
oxygen p e r hour and male A s c a r is u t i l i z e d s l i g h t l y more.
Davey (1937, 1938) showed t h a t i n com plete absence
o f oxygen v a r io u s sheep nem atodes d ie d w ith in 48 h o u rs .
I n o t h e r e x p e rim e n ts , movement o f t h e worms (u n d e r hydrogen)
remained norm al a t f i r s t , bu t l a t e r became slow and s p a s ­
modic; u s u a l l y c e a sin g e n t i r e l y a f t e r f o u r h o u rs .
I f a ir
was a d m itte d , t h e power o f norm al movement was q u ic k ly
(a p p ro x im a te ly 15 m inu tes) r e g a i n e d .
A f t e r f i f t e e n h o u rs
most worms f a i l e d to r e g a i n movement; b u t t h r e e which d id
-8 -
r e c o v e r , d i d n o t b e g in t o move u n t i l f i v e h o u rs a f t e r
rem oval o f t h e h ydrogen, and th e n movements were slow and
i n f r e q u e n t u n t i l t h e end o f t h e i r l i f e .
Hookworms s u p p ly a v e ry i n t e r e s t i n g e x c e p tio n to
any assu m p tio n t h a t i n t e s t i n a l p a r a s i t e s a r e c h i e f l y an­
a e ro b ic .
i n 1931, W ells observed t h a t hookworms sucked
blood alm o st c o n tin u o u s ly and he e s tim a te d t h a t i n 24 hours
one worm would ta k e up 0 .8 4 c c . o f t h e h o s t ’ s b lo o d . T h is
blood p a ssed th ro u g h th e gut to o r a p i d l y f o r d i g e s t i o n . He
a l s o o b se rv e d t h a t oxygen could be ab so rb e d by th e worms,
and he su g g e ste d t h a t t h e blood may s e rv e a r e s p i r a t o r y
fu n c tio n .
Harwood and Brown (1933) showed t h a t hookworms
un d er a e r o b ic c o n d i t i o n s u t i l i z e oxygen p e r gram o f body
w eigh t a t a r a t e t e n tim e s g r e a t e r t h a n does A s c a r i s , a
worm t h a t l i v e s i n th e i n t e s t i n a l lumen and does not suck
b lo o d .
Von Brand and O tto (1938) found t h a t t h e glycogen
c o n te n t o f th e hookworm, Ancylostoma caninum was on t h e
a v e ra g e abou t 1 .5 p e r c e n t o f t h e t o t a l w eight o f f r e s h
worms, whereas i n A s c a r i s i t I s about 5 .5 p e r c e n t . They
a l s o found t h a t t h e t o t a l glycogen c o n te n t o f t h e worms I s
a p p a r e n t l y unchanged by s h o r t p e r io d s o f s t a r v a t i o n o r a l i ­
m entary hy perg ly cem ia o f the h o s t .
They a ig g e s te d t h a t t h e
b lo o d -s u c k in g h a b i t s o f th e worm may p e rm it an o x i d a t i v e
ty p e o f m etab o lism , and s in c e a c o n s t a n t food su p p ly i s a v a i l a b l e f o r t h e same re a s o n , t h e s e worms do n o t need to
-9 -
s t o r e l a r g e amounts o f m a t e r i a l t o be u sed b o th f o r n u t r i ­
t i o n and as a so u rc e o f oxygen.
S ta n n a rd , McCoy and L a tc h f o rd (1938) conducted
an i n v e s t i g a t i o n o f th e m etabolism o f a t i s s u e p a r a s i t e , t h e
la rv a e of T r lc h in e lla s p i r a l i s .
The environm ent o f t h e s e
l a r v a e i s d e c id e d ly d i f f e r e n t from t h a t o f i n t e s t i n a l p a r a ­
site s .
The l a r v a e , which a re e n c y s te d i n m uscle t i s s u e ,
e x i s t n a t u r a l l y i n an environm ent which t h e s e auth ors con­
s i d e r e d to be u n d o u b ted ly a e r o b i c .
Such l a r v a e rem ain f o r
many y e a r s i n s i t u , so i t seems th e y must a s s i m i l a t e food
d e r iv e d from th e h o s t t i s s u e s .
McCoy, Downing, and
Van V oorhis (1941) fe d r a d i o a c t i v e phosphorus ( a s b a s i c
sodium p h o sp h ate) t o t r i c h i n i z e d r a t s and d em o n stra te d i t s
a b s o r p t i o n by t h e en c y ste d worms as e a r l y a s two h o u rs a f t e r
fe e d in g .
The phosphorus re ach ed a maximal c o n te n t i n t h e
l a r v a e by th e f o u r t h day a f t e r which a g ra d u a l d e c l i n e o c cu rred .
T h is a b s o r p t i o n and l o s s o f r a d iA c t iv e phosphorus
f
d id n o t fo llo w t h e same c o u rse i n th e l a r v a e as i t did i n
t h e m uscles o f t h e h o s t .
These a u th o r s su g g e ste d t h a t t h i s
exchange^of phosp h ate io n s th ro u g h t h e c y s t w a ll i n d i c a t e d
t h a t th e l a r v a e were un derg o in g a c t i v e m etab o lism .
S ta n n a rd , McCoy, and L a tc h f o rd found a f a i r l y
h ig h r a t e o f r e s p i r a t i o n a s measured by oxygen consum ption
in tric h in a la rv a e .
A f e r m e n ta tiv e ty p e o f m etabolism was
e v id en c ed by ca rb o n d io x id e p r o d u c tio n u n d er a n a e ro b ic
-1 0 -
c o n d i t i o n s , b u t t h e a e r o b i c m etabolism was c o n s id e r a b ly
g r e a te r th a n th e fe rm e n ta tiv e .
The r a t e s , however, f o r
b o th ty p e s o f m etabolism were re m ark ab ly c o n s ta n t i n numer­
o u s e x p e rim e n ts w ith d i f f e r e n t l o t s o f l a r v a e . The r e s p i r a ­
t o r y q u o t i e n t was s l i g h t l y above one.
These a u th o r s be­
l i e v e d t h a t f e r m e n ta tio n o c c u rre d un d er a e r o b i c c o n d i t i o n s ,
a s w e ll a s d u rin g a n a e r o b i o s i s .
They c o n s id e re d i t ffim prob-
a b l e t h a t a w e ll-d e v e lo p e d r e s p i r a t o r y mechanism s i m i l a r
i n im p o rta n t r e s p e c t s t o th o s e o f an a e r o b ic organism would
be p r e s e n t i n an organism e x i s t i n g s o l e l y by an a n a e ro b ic
mechanism.
On t h e o th e r hand, t h e r e i s a d e f i n i t e fe rm e n ta ­
t i o n d i f f e r i n g i n many c h a r a c t e r i s t i c s from t h a t o f m uscle
o r o t h e r nem atodes, b u t a p p a r e n tly c a p a b le o f s u p p o rtin g
t h e organism d u rin g p e r io d s o f a n a e r o b i o s i s . "
Io d o a c e ta te ,
a t a c o n c e n t r a t i o n known t o i n h i b i t m uscle g l y c o l y s i s r a p i d l y ,
was o n ly a slow i n h i b i t o r o f th e f e r m e n ta tio n o f t h e s e l a r v a e .
T here was a somewhat com parable d i f f e r e n c e u s in g i o d o a c e t a m ide.
T h is seemed t o be a somewhat b e t t e r i n h i b i t o r o f
f e r m e n t a t io n i n t h e l a r v a e , but i t was v e ry slow i n com parison
w ith i t s e f f e c t s on a l c o h o l i c f e r m e n ta tio n o r muscle g l y c o l y ­
sis.
At t h e same tim e , u n l i k e m uscle o r y e a s t m etab olism ,
t h e r e s p i r a t i o n was more s e n s i t i v e t h a n th e f e r m e n ta t io n .
Sodium f l u o r i d e showed an even g r e a t e r d i f f e r e n c e t h a n d id
t h e iodo compounds.
I t i s well-known t h a t cy an id e i n h i b i t s th e r e s p i r a -
-1 1 -
t i o n o f most a e r o b i c o rg a n ism s, w hile t h e a n a e ro b ic system
i s n o t s e n s i t i v e to low c o n c e n t r a t i o n s o f t h i s p o is o n .
The
W arb u rg -K e ilin Cytochrome system which i s c y a n i d e - s e n s i t i v e
has a v ery wide d i s t r i b u t i o n i n a e r o b ic o rg a n ism s. T r i c h i n e l l a
l a r v a e were found t o be very s e n s i t i v e to c y a n id e .
e f f e c t was r e v e r s i b l e .
T h is
Carbon monoxide, while n o t c o n s i d e r ­
ed t o be a s i n h i b i t o r y t o c e l l u l a r r e s p i r a t i o n as c y a n id e ,
w i l l a t h ig h t e n s i o n s i n h i b i t r e s p i r a t i o n in v o lv in g th e
cytochrom e sy stem .
T h is i n h i b i t i o n i s r e v e r s e d by l i g h t .
The r e s p i r a t i o n of T r i c h i n e l l a l a r v a e was s t i m u l a t e d r a t h e r
th a n i n h i b i t e d by ca rb o n monoxide.
While many t i s s u e s a re
i n s e n s i t i v e t o t h i s p o is o n , o n ly s k e l e t a l and c a r d i a c m uscle
( f r o g and r a t ) a r e known to be s tim u la te d (Fenn and Cobb
1 9 3 2 a ,b ) .
S ta n n a r d , McCoy, and L a tc h fo rd p o in te d o u t t h i s
s t r i k i n g s i m i l a r i t y between a p a r a s i t e o f muscle t i s s u e and
th e tis s u e i t s e l f .
They concluded t h a t th e a e r o b ic mechan­
ism o f t h e s e p a r a s i t e s i s r e l a t i v e l y s i m i l a r to t h a t o f
mammalian m uscle, w hile t h e a n a e r o b ic f e r m e n ta tio n i s r e l a t i v e ­
ly d is s im ila r.
Cyanide o r l a c k o f oxygen caused t h e l a r v a e
t o become i n a c t i v e , a lth o u g h th e y were a b le to l i v e f o r a
week o r l o n g e r , u n d e r a n a e ro b ic c o n d i t i o n s .
Thus, i n t h e i r
norm al environm ent i n m uscle t i s s u e , T r i c h i n e l l a l a r v a e p ro b ­
a b ly e x i s t p r i n c i p a l l y by an a e r o b ic m etabo lism .
I n 1934, F ried h eim measured m a n o m e trlc a lly th e
-IE -
r e s p i r a t o r y a c t i v i t i e s o f e g g s , p l e r o c e r c o i d s and a d u l t s
o f D lp h v llo b o th riu m la tu m .
He found t h a t a l l s ta g e s ab­
s o rb oxygen and to an in c r e a s e d d e g re e i n th e p re s e n c e o f
g lu c o s e .
An atm osphere o f 95 p e r c e n t carbon monoxide
d id n o t show any i n h i b i t o r y e f f e c t on t h i s p a r a s i t e . P o t a s ­
sium cy a n id e i n h i b i t s r e s p i r a t i o n o f th e a d u l t in c o m p le te ly
even i n M/100 c o n c e n t r a t i o n , w hereas M/100 c o n c e n t r a t i o n
i n h i b i t e d a b s o r p tio n i n eggs and l a r v a e .
F ried h elm s t a t e d
t h a t th e a d u l t showed two a b s o r p t i o n bands of Cytochrome C.
The g e n e r a l c o n c lu s io n s t h a t can be drawn from
th e i n v e s t i g a t i o n s o f t h e s e v a r io u s p a r a s i t e s are t h a t most
o f them p o s s e s s an a n a e ro b ic mechanism f o r m etabolism , which
s e r v e s them f o r g r e a t e r o r l e s s e r p e r io d s o f oxygen l a c k ;
y e t none o f them seem i n j u r e d when p la c e d u n d e r a e r o b ic con­
d i t i o n s , and many o f t h e s e p a r a s i t e s are a b le to ta k e oxygen
from t h e en v iro n m en t.
Thus t h e y a r e q u i t e d e f i n i t e l y n o t
t r u e an a e ro b e s i n th e b a c t e r i o l o g i c a l s e n s e .
Von Brand
(1934b) found t h a t f o r 1 .3 9 gm glycogen u t i l i z e d by 100
grams o f A s c a r is d u rin g 24 h o u rs a n a e r o b i o s i s , 0 .7 1 gm c a r ­
bon d i o x i d e , 0 .2 2 gm v a l e r i c a c i d , end 0 .0 2 gm l a c t i c a c id
were p rod uced.
He c o n s id e re d t h a t an e q u a tio n m ight be
d e r iv e d when o t h e r end p ro d u c ts as y e t unknown a r e d e te rm in e d .
He a l s o found t h a t a f e r m e n ta tiv e m etabolism p e r s i s t e d i n
A s c a r is u n d er a e r o b ic c o n d i t i o n s .
O th er i n t e s t i n a l p a r a s i t e s (fla tw o rm s) gave r i s e
-1 3 -
t o end p r o d u c ts s i m i l a r i n amount, b u t h ig h e r f a t t y a c id s
were more p r e v a l e n t .
L a c t i c a c i d and s u c c in i c a c id o c c u rj
.
red i n th e e n d -p ro d u c ts when M oniezia expanse was s t u d i e d ,
(von B rand, 1929, 1933; A lt and T i s c h e r , 1 9 3 1 ).
Weinland
and von Brand (1926) showed a s i m i l a r m etabolism f o r
F a sc io la h e o a tlc a .
The stu d y o f t h e n u t r i t i o n o f e n to z o a i s q u i t e
o b v io u s ly a f u n c t i o n o f th e s tu d y o f and a tte m p ts a t i n
v itr o c u ltu re of in te rn a l p a ra s ite s .
C om paratively l i t t l e
i s known o f t h e food r e q u ir e m e n ts o f t h e s e a n im a ls . McCoy
(19 29a,b) found t h a t hookworm l a r v a e would develo p t o t h e
i n f e c t i v e s ta g e w ith j u r e c u l t u r e s o f b a c t e r i a as t h e i r
o n ly food s o u rc e .
However, he does " n o t e x c lu d e t h e p o s s i ­
b i l i t y t h a t o t h e r s u b s ta n c e s th a n b a c t e r i a may s e rv e as
food f o r hookworm l a r v a e " .
Lapage (1933) r e a r e d v a r i o u s
sheep and r a b b i t nematode l a r v a e on pure c u l t u r e s o f
B a c illu s c o l l .
G la s ^ e r and S t o l l (1 9 3 8 a ,b ) re a r e d t h e f r e e -
l i v i n g s ta g e s o f t h e sheep stom ach worm Haemonchus c o n t o r t u s
i n s t e r i l e m edia.
These r e p r e s e n t some o f th e more impor­
t a n t p a p e rs d e a lin g w ith th e c u l t i v a t i o n o f f r e e - l i v i n g
s t a g e s o f p a r a s i t i c worms.
H o e p p li, Feng and Chu (1938) r e ­
viewed a tte m p ts t o c u l t u r e h e lm in th s i n a r t i f i c i a l m edia.
F r e e - l i v i n g and f a c u l t a t i v e - p a r a s i t i c nem atodes were d i s ­
cussed a s w e ll as f r e e - l i v i n g and p a r a s i t i c s ta g e s o f o b l i ­
g a to ry p a r a s i t e s .
-1 4 -
The p r i n c i p a l q u e s tio n i s not concerned w ith th e
n u t r i t i o n d u rin g f r e e - l i v i n g s t a g e s , but a f t e r th e p a r a s i t e
has s e t t l e d w i th in t h e h o s t .
The stu d y o f e x c r e t o r y p ro d u c ts
and th e d e m o n s tra tio n t h a t glycogen i s used d u r in g a n a e ro b ic
l i f e g iv e s some i m p l i c a t i o n o f food s o u rc e .
F lu r y (1912)
s t u d ie d t h e e x c r e t o r y p ro d u c ts o f A s c a r i s , b u t f a i l e d to
f i n d ev id e n c e t h a t t h e u s u a l e n d - p o in ts o f p r o t e i n m etab o l­
ism , such as u r e a , u r i c a c i d , c r e a t i n i n , and th e p u r in e
bases r e s u lte d .
however.
Ammonium s a l t s and f a t t y a c id s d id o c c u r
M u e lle r (1 9 2 9 ), on t h e b a s i s o f m o rp h o lo g ic a l
e v id e n c e , su g g e ste d t h a t f a t a s w e ll a s c a rb o h y d ra te m ight
be u t i l i z e d by A s c a r i s .
Von B rand,how ever, c o n s id e re d f a t s
to be e x c r e t o r y p ro d u c ts r a t h e r th a n food r e s e r v e s .
If
th e y a r e e x c r e to r y p r o d u c ts , i t would be ex p e cted t h a t , due
to g r a d u a l a c c u m u la tio n , o l d e r worms would have a g r e a t
amount p r o p o r t i o n a l l y .
Smorodinzew and B ^beschin (1935)
made a n a ly s e s o f a n t e r i o r , m id d le , and p o s t e r i o r p o r t i o n s
o f T a e n ia s a g l n a t a .
They found t h a t t h e a n t e r i o r r e g io n s had
th e h ig h e s t p e r c e n ta g e v a lu e f o r l i p o i d s . Somewhat i n su p p o rt
of von B rand, P e t e r s (1936) h as s u g g e s te d t h a t t h e m eta­
b o l i c r a t e i s p ro b a b ly h ig h e r i n t h e younger segm ents, t h a t
o l d e r segm ents a r e m ostly " e g g - s a c s " , and t h a t p o s s i b l y f a t
d e p o s i t i o n i s o n ly tem p o rary .
Under c o n d i t io n s o f s t a r v a t i o n ,
von Brand was u n a b le t o show a m a t e r i a l d e c re a s e o f f a t s ,
a lth o u g h glycogen was u t i l i z e d .
From t h e s t a n d p o i n t o f nem atodes, some d a t a on
n u t r i t i o n have ac cu m u lated .
As n o te d e a r l i e r , V e ils had
su g g e s te d t h a t t h e blood p a s s e s th ro u g h t h e g u t o f hook­
worms to o r a p i d l y f o r d i g e s t i o n , and he s u g g e ste d t h a t
t h e s e worms u t i l i z e d i f f u s i b l e s u b s ta n c e s i n th e plasma
f o r fo o d .
S ta n n a r d , McCoy,and L a tc h f o rd (1938) p o in te d
o u t t h a t T r i c h i n e l l a s p i r a l i s l a r v a e p ro b a b ly a s s i m i l a t e
food d e r iv e d from h o st t i s s u e s , s i n c e th e y rem ain f o r many
years in s i t u .
I n t h e i r e x p e rim e n ts , th e a d d i t i o n o f g lu ­
c o s e , g ly co g en , l a c t a t e , p y r u v a te , s u c c i n a t e , hexose d i ­
p h o s p h a te , serum, o r Douglas* medium d id not cau se any s i g ­
n i f i c a n t r i s e i n oxygen consum ption.
A ckert and W hitlock (1935) and A ck e rt and Freeman
(1936) have shown t h a t A s c a r l d i a l i n e a t a . a p a r a s i t e o f
c h ic k e n s , ta k e s n ou rishm en t from th e food i n t h e i n t e s t i n a l
lumen o f t h e h o s t , r a t h e r th a n from t h e e p i t h e l iu m . Normally
fe d c o n t r o l s were compared w ith b i r d s fed by v a r i o u s t i s s u e
and p a r e n t e r a l i n j e c t i o n s .
Where no c h ic k e n food was p r e s e n t
i n th e fowl i n t e s t i n e , th e worms made p r a c t i c a l l y no grow th.
H oeppli (1933) review ed th e work on e x t r a - i n t e s t i n a l
d ig e stio n .
Much o f t h i s work had been done w ith forms having
l a r g e b u c c a l c a p s u le s , b u t i t seems p ro b a b le t h a t l i q u e f a c ­
t i o n o f t h e h o s t t i s s u e s p ro v id e s e le m e n ts t h a t a r e u t i l i z e d
f o r food by c e r t a i n nem atodes.
Weinland and von Brand (1926) agreed w ith an
e a r l i e r w orker, M u ller (1 9 2 3 ), t h a t F a s c i o l a h e u a t l c a fe e d s
on t i s s u e e le m e n ts and Inflam m atory ex u d a te s r a t h e r th a n
on b lo o d .
Tapeworms, h aving no a lim e n ta r y c a n a l , fe ed by
o sm o sis.
T h e r e f o r e , t h e y must u t i l i z e foods which can
n o rm a lly be absorbed th ro u g h t h e i n t e s t i n a l w a ll.
Most
i n v e s t i g a t o r s seem to t h i n k t h a t g lu c o s e i s one o f th e
p r i n c i p a l foods u t i l i z e d .
Wardle (1937) showed t h a t about
32 p e r c e n t o f the dry w eight o f M oniezia i s p o l y s a c c h a r i d e ,
and he s t a t e d t h a t "G lucose seems d e f i n i t e l y th e one c a rb o ­
h y d r a te re q u ire m e n t o f tapeworms; t h e anlmo acid re q u ire m e n t
may be e q u a ll y sim p le ; t h e r e may be no f a t r e q u ir e m e n t ."
S a l i s b u r y and Anderson (1939) found 30 p e r c e n t o f t h e d ry
weight o f t h e l a r v a e o f T a e n ia t a e n i a e f o r m l s t o be g ly co g en .
Smorodinzew and E eb esch in (1933, 1935, and 1936) found a
somewhat e q u iv a l e n t amount o f o r g a n ic m a t e r i a l a f t e r removal
o f l i p o i d and p r o t e i n components i n t h e i r i n v e s t i g a t i o n s on
T aen ia solium and 3Lt s a g l n a t a .
They d id not r e f e r t o t h i s
p o r t i o n a s p o l y s a c c h a r i d e , b u t i t seems l i k e l y t h a t i t I s .
S ch o p fe r (1932) working w ith C v s tlc e r c u s t e n u i c o l l i s
o f sh e e p , found t h a t t h e c y s t i c f l u i d was poor i n p r o t e i n s
and r i c h i n c h l o r i d e s and c a r b o h y d r a te s , and he concluded
t h a t th e l a t t e r r a t h e r t h a n th e p r o t e i n s a re used a s a food
s o u rc e by th e p a r a s i t e .
Lemaire and R ib e re (1935) d is c o v e r e d
17-
a g l y c o l y t i c enzyme i n h y d a tid f l u i d , and t h i s might sug­
g e s t t h a t l a r v a l t a p e s u t i l i z e a m etabolism based on g ly co ­
gen d e c o m p o s itio n .
A ttem pts a t c u l t u r i n g en to z o a a s m entioned e a r l i e r ,
have g iv e n l i t t l e In the way o f a p o s i t i v e answer t o th e
q u e s t i o n o f food r e q u ir e m e n ts o f t h e forms s t u d i e d , a lth o u g h
i t seems p ro b a b le t h a t f a c t o r s o th e r th a n t h e food I t s e l f
p ro v id ed t h e p r i n c i p a l stu m b lin g b lo c k s .
P a r a s i t e s o f t h e i n t e s t i n e and b i l e - d u c t have
been m ain tain ed a l i v e f o r v a ry in g numbers o f d a y s .
The
b a s i s f o r most o f th e s e media was some ty p e o f i s o t o n i c s a l t
s o l u t i o n , a lth o u g h i t i s o f i n t e r e s t to n o te t h a t B a y lis
( 1 9 2 2 ) ,i n a d i s c u s s i o n o f th e c o l l e c t i o n and p r e s e r v a t i o n o f
p a r a s i t i c worms, s t a t e d t h a t some a u t h o r i t i e s b e l i e v e t h a t
t a p - w a te r i s b e t t e r f o r washing i n t e s t i n a l nem atodes p r e p a r a ­
t o r y t o f i x a t i o n t h a n i s any s a l t s o l u t i o n ; and M eg g itt
( 1 9 2 4 ) ,i n a s i m i l a r d i s c u s s i o n of te c h n iq u e f o r tapew orm s,
s a id t h a t s a l t s o l u t i o n should n ev e r be u s e d .
A lthough i n ­
t e s t i n a l p a r a s i t e s have rem ained a l i v e f o r a number o f days
i n Normal s a l t s o l u t i o n s , (B unge,1803; H a l l , 1917; V ogel,
1934) food s o u rc e s must be a v a i l a b l e f o r any s a t i s f a c t o r y c u l ­
tiv a tio n o f liv in g m a te ria l.
Lonnberg, (1892) m a in ta in e d T ria e n o n h o ru s n odu lo su s
from th e i n t e s t i n e o f Esox ov er a month u s in g a v e ry d i l u t e
-1 8 -
p e p s in -p e p to n e s o l u t i o n .
Magath (1919) m a in ta in e d
C am allanus am ericanu s from t u r t l e i n t e s t i n e ab o u t two
months u s in g W i tt e ’ s pep to n e and g e l a t i n e and t u r t l e blood
ag ar.
Meier (1913) m a in ta in e d Distomum t e r e t i o o l l e abou t
t h r e e weeks u s in g c o n c e n tr a te d Locke s o l u t io n w ith p e p to n e .
S tu n k a rd (1930) k e p t m e t a c e r c a r i a e o f C rv n to c o tv le l i n g u a
a l i v e s i x days a t body te m p e r a tu r e , 14 days a t room tem per­
a t u r e i n an i s o t o n i c s a l t - d e x t r o s e medium to which d i g e s t e d
v e a l e x t r a c t was added.
I n 1932, he k e p t specim ens o f
C ren id o b o th riu m lS n n b e r e i a l i v e 32 d ays I n i s o t o n i c s a l t d e x t r o s e s o l u t i o n s v a r i o u s l y m o d ifie d w ith v e a l d i g e s t .
Adam (1932) m a in ta in e d A s c a r is suum up t o 22 days i n Tyrode
so lu tio n .
S tunkard (1940) r e a r e d t h e tre m a to d e , P l a e l t u r a
n a rv a from t h e m e t a c e r c a r i a l s ta g e t o s e x u a l m a t u r i t y i n
v e a l d i g e s t - s a l t - d e x t r o s e media to which l i v e y e a s t was
added.
H o e p p li, Peng, and Chu (1938) m a in ta in e d th e nema­
to d e s P h y s a lo n te r a c l a u s a and T h e la z ia c a l l i c a e d a f o r t e n to
tw elv e days i n normal s a l i n e t o which d i l u t e d h o rse serum
was added.
These a u th o r s kept C lo n o rc h is s i n e n s i s i n d i l u t e d
h o rs e serum Tyrode s o l u t i o n a maximum o f f i v e m onths.
Mendelsohn (1935) k e p t v e ry m inute l a r v a e o f T aenia
t a e n i a e f o r m i s a l i v e f o r 32 days i n b a la n c e d s a l t s o l u t i o n ,
c h ic k embryo e x t r a c t , and h o rs e serum.
Larvae o f M u ltic e n s
s e r i a l l s from subcu taneo us t i s s u e o f r a b b i t were m a in ta in e d
-1 9 -
20 days In norm al s a l i n e and f r e s h serum , C o u t e l e n ,(1 9 2 9 ).
ftch ln ococcus g ra n u lo s u s rem ained a l i v e two weeks i n h o rs e
serum and h y d a tid f l u i d to which serum o r a s c i t e s f l u i d o r
a l c o h o l i c e x t r a c t o f sheep l i v e r was added.
H o e p p li, Feng,
and Chu r e p o r te d t h a t p ig A s c a r is l a r v a e were k e p t a l i v e one
week I n d i l u t e d h o rs e serum and d e b r i s o f f r e s h l i v e r .
They
k e p t l a r v a e of T a e n ia t a e n l a e f o r m i s and Snargenum e r l n a c e l
( l a r v a o f D ip h y llo b o th riu m e r i n a c e i ) a l i v e 9 - 1 8 days i n
v a r i o u s s e r a , t i s s u e e x t r a c t s , and b r o th s o l u t i o n s .
Growth o r changes a p p ro x im a tin g growth h a s been r e ­
p o rte d f o r p a r a s i t e s o f v e r t e b r a t e s i n o n ly a few c a s e s .
G la s e r and S t o l l (1938) d em o n strated growth and t i s s u e changes
i n th e sheep nematode Haemonchus c o n t o r t u s d u rin g t h e f r e e l i v i n g l a r v a l s t a g e s and up to th e I n f e c t i v e s t a g e . A c k e rt,
Todd, and Tanner (1938) d em o n stra te d grow th i n p a r a s i t i c l a r v a e
o f A 3 c a rld la l l n e a t a i n v i t r o .
These worms had begun t h e i r
developm ent i n t h e c h ic k e n i n t e s t i n e and were th e n t r a n s f e r r e d
t o c u l t u r e m edia.
C a l l o t (1939) found t h a t some developm ent
o c c u rre d i n r e p r o d u c tiv e o rg a n s o f t h e s t r i g i e d P o s th o d i n l o s tomum c u t i c o l a i n p h y s i o l o g i c a l s a l i n e .
Stunkard (1940)
r e a r e d m e ta c e r c a r ia e o f P l a g i t u r a n arv a t o s e x u a l m a t u r i t y i n
a b a c t e r i a - f r e e medium.
F erg uso n (1940) got egg p ro d u c tio n
i n v i t r o I n a d u l t s o f the tre m a to d e P osthodlnlostom um minimum,
which had been r e a r e d from m e t a c e r c a r i a e .
The r e f e r e n c e s c i t e d c o n s t i t u t e th e more im p o rta n t
-2 0 -
i n v i t r o c u l t u r e e x p e rim e n ts r e p o r t e d f o r i n t e s t i n a l and
t i s s u e p a r a s i t e s from th e s t a n d p o i n t o f l o n g e v i t y and
grow th.
Blood p a r a s i t e s such a s S ch istosom a and m i c r o f i l ­
a r i a have been m a in ta in e d f o r weeks by v a r io u s a u th o r s on
such media as v a r i o u s s e r a , d e f i b r i n a t e d b lo o d , and coagu­
l a t e d b lo o d , (H o e p p li, Feng, and Chu, 1 9 3 8 ), b u t such d a t a
a re n o t p a r t i c u l a r l y p e r t i n e n t t o t h e problem a t hand.
OTHER CONSIDERATIONS OF HELMINTH PHYSIOLOGY
F undam entally a s s o c i a t e d w ith t h e developm ent o f
a p a r a s i t i c mode of l i f e a r e th e a d a p t a t i o n s t h a t have oc­
c u r re d to meet s u c c e s s f u l l y th e e x i g e n c ie s o f th e e n v ir o n ­
ment.
M o d if ic a tio n s i n food h a b i t s , r e s p i r a t i o n , o sm o tic
and pH r e l a t i o n s h i p s , s e c r e t i o n s , and r e p r o d u c t i o n have o c ­
cu rred .
Some o f t h e s e a lr e a d y have been r e f e r r e d t o and
o t h e r s w i l l be m entioned b r i e f l y .
To m a in ta in th e s t a t u s quo o f t h e b i o t i c p o t e n t i a l ,
p a r a s i t e s have undergone numerous changes i n r e p r o d u c t i v e
c a p a c itie s.
lin e d .
T hese a r e well-known and on ly need to be o u t ­
Host s p e c i f i c i t y and t h e co m p lex ity o f l i f e c y c le a r e
m ajor f a c t o r s a f f e c t i n g t h e s e c h a n g e s.
most u n b e l i e v e a b le numbers o f e g g s.
i n th e o n e -h o st l i f e c y c l e s .
P a r a s i t e s produce a l ­
T his f e a t u r e o c c u rs even
F a c t o r s which i n s u r e f e r t i l i z a ­
t i o n o f t h e eggs a r e h e rm a p h ro d itism , and s e l f - f e r t i l i z a t i o n .
M u l t i p l i c i t y o f segments b e a r in g gonads, and i n some c a s e s
-2 1 -
d u p l i c a t i o n i n t h e same segm ent i s a n o th e r method f o r v a s t l y
I n c r e a s i n g p r o d u c tio n .
P a r t i c u l a r l y smong th e fla tw o rm s,
a s e x u a l r e p r o d u c t io n among l a r v a l s t a g e s i n c r e a s e s t h e number
of p o te n tia l a d u lt p a r a s ite s .
S e c r e t i o n s o f e n to z o a seem i n p s r t t o be a r e s u l t
of p a ra sitism .
B oring g la n d s among tre m a to d e l a r v a e a r e adap­
t a t i o n s t o t h e mode o f e x i s t a n c e .
H oeppli (1933) review ed
e x t r a - i n t e s t i n a l d i g e s t i o n in p a r a s i t e s .
T his f e a t u r e may be
a c a r r y - o v e r from days b e fo re p a r a s i ti s m d e v e lo p e d , s in c e
among t h e T u r b e l l a r i a n s e x t r a - i n t e s t i n a l d i g e s t i o n i s common.
Weinland (1903) r e p o r t e d ev id en c e o f an ti-en z y m e s i n A s c a r i s .
McCoy i n h i s rev iew (1935) m entioned t h a t H am ill (1906)
d e m o n stra te d an a n t i - t r y p t i c enzyme i n w a t e r - e x t r a c t s o f
A s c a r i s , and i n 1907, F e t t e r h o f d em o n strated a n ti-e n z y m e s i n
T a e n ia s a g l n a t a .
Harned and Nash (1932) found an a n t i ­
t r y p s i n , and a n t i - p e p s i n , and a p r o t e a s e i n a w ater e x t r a c t
o f A sc a ris.
Sang (1938) found a n t i - p r o t e o l y t i c enzymes I n 8
w ate r e x t r a c t o f A s c a r i s .
Schw artz (1921) d em o n strated a n t i -
c o a g u lin s i n A s c a r is and Haemonchus e x t r a c t s .
Lemaire and
R ib e r e , a s p r e v io u s l y m entioned , d is c o v e r e d a g l y c o l y t i c en­
zyme i n h y d a tid f l u i d .
I t i s common knowledge t h a t b alan ced s a l t s o l u t i o n s
a r e fu n d am en tal and t h a t s e r a and o t h e r body f l u i d s show a
marked s i m i l a r i t y t o sea w ater i n s a l t c o n te n t and r a t i o s .
-2 8 -
That t h e s a l t r a t i o i s b a s i c i s shown by t h e e f f e c t o f ab­
normal s a l t s o l u t i o n s on h e a r t b e a t , o r abnorm al se a w ater
on developm ent o f eggs o f m arine a n im a ls .
B alan ce d s a l t s o l u t i o n s have been u sed as th e b a s i s
f o r most of t h e media used i n a tt e m p t s t o c u l t u r e jjn v i t r o .
T h is i s p a r t i c u l a r l y l o g i c a l w ith re g a rd t o t i s s u e p a r a s i t e s ,
which u n q u e s tio n a b ly w i l l be i n an environm ent s u b je c t t o
m inim al f l u c t u a t i o n s a s compared w ith th e i n t e s t i n a l lumen.
Davey (1938) showed t h a t com p lete absence o f c e r t a i n s a l t s
from t h e en vironm ent r e s u l t e d i n d e a th f o r v a r io u s nematode
p a r a s i t e s o f the sheep a lim e n ta r y c a n a l .
Some f l u c t u a t i o n i n
t h e s a l t r a t i o s d i d n o t seem t o be h a rm fu l, however.
H ydrog en -ion c o n c e n t r a t i o n i s a f a c t o r th a t a l s o w i l l
undergo f a r l e s s change f o r t i s s u e p a r a s i t e s t h a n f o r th o s e
l i v i n g i n t h e a li m e n t a r y c a n a l .
McCoy (1930) s t u d i e d th e
a f f e c t of t e m p e r a tu r e , h y d ro g e n -io n c o n c e n t r a t i o n , and oxygen
t e n s i o n on t h e developm ent o f eggs and l a r v a e o f Ancvlostoma
canlmum.
He concluded t h a t t h e s e s t a g e s a r e n o t s e n s i t i v e to
wide v a r i a t i o n s o f su ch e n v iro n m e n ta l f a c t o r s , and, i n 1935,
he a p p lie d t h i s c o n c lu s io n t o p a r a s i t e s more g e n e r a l l y .
H o e p p li, Feng, and Chu (1938) s t a t e t h a t "Most o f t h e more
r e c e n t p u b l i c a t i o n s em phasize t h e im p o rtan ce o f a p r o p e r l y
a d j u s t e d pH o f t h e medium.
A pH abo ut 7 .3 - 7 .6 seems i n gen­
e r a l t o be t h e b e s t . A pH beyond 8 r e s u l t s u s u a l l y i n a r a p id
d e a th o f th e h e lm in th s ; on t h e o t h e r hand, a pH ra n g in g
between 5 .4 and 7 i s n o t d e t r i m e n t a l f o r some s p e c ie s a s ,
f o r example, f i l a r i f o r m l a r v a e o f S tro n g y lo ld e s f u l l e b o r n i
(Chung, 1 9 3 6 )" .
Davey (1937) found f o r nem atodes o f th e
sheep i n t e s t i n e t h a t v a r i a t i o n s o f 3 .2 - 9 .0 and 4 .4 - 9 .0
were not l e t h a l when th e worms were s u b je c te d c o n s t a n t l y to
such h y d ro g e n -io n c o n c e n t r a t i o n s , a l t h o u g h a t t h e a c id end
o f t h e s c a l e p a r t i c u l a r l y a tim e elem ent was in v o lv e d .
* S tu nkard (1932) showed t h a t a pH o f 7 .3 was optimum f o r
growth w ith C re rld o b o th riu m l o n n b e r g i . I n g e n e r a l , a pH
between 7 and 8 i s p ro b a b ly a f a i r l y s a f e and e f f e c t i v e
ran g e a c c o rd in g to th e concensus o f o p in io n .
ACKNOWLEDGMENT
I w ish t o ta k e t h i s o p p o r tu n ity t o e x p re s s a p p re ­
c i a t i o n f o r th e a i d and s u g g e s tio n s r e c e i v e d from Dr. M. R,
C u r t i s , Dr. W. F. D unning, and t h e l a t e Dr. F. D. B u llo c k
o f t h e I n s t i t u t e o f Cancer R e s e a rc h , Columbia U n i v e r s i t y i n
s t a r t i n g and m a in ta in in g th e tapeworm, T ae n ia t a e n l a e f o r m i s .
i n l a b o r a t o r y a n im a ls ; and f o r t h e i n t e r e s t and s u p p o rt o f
members o f th e B iology F a c u l t y a t Brooklyn C o lle g e .
S p e c ia l
th a n k s a r e due members o f t h e E io lo g y F a c u lty a t New York
U n i v e r s i t y , U n i v e r s i t y H e ig h ts , f o r i n t e r e s t and s u g g e s tio n s ,
and p a r t i c u l a r l y t o P r o f e s s o r D an iel Ludwig f o r h e lp and
c o n s t r u c t i v e c r i t i c i s m i n t h e p r e p a r a t i o n o f t h e m a n u sc rip t
-2 4 -
and t o P r o f e s s o r Horace W. S tunk ard u n d er whose d i r e c t i o n
t h i s stu d y was made and whose c o n s id e r e d g u id an ce was i n ­
v a l u a b l e a t every s t e p .
PROBLEM
Inasmuch as many p a r a s i t e s a r e m easurably p a th o ­
g e n ic , a com plete knowledge o f t h e i r b io lo g y i s needed t o
circ u m v en t t h e i r a c t i v i t i e s most e f f i c i e n t l y .
C om paratively
l i t t l e i s known o f t h e p h y s io lo g y o f i n t e s t i n a l and t i s s u e
p a r a s i t e s and s in c e c o n t r o l l e d i n vivo ex p e rim e n ts p r e s e n t
obvious d i f f i c u l t i e s , an a tte m p t lias been made to d e v is e
methods f o r t h e i n v i t r o m ain ten an ce o f some common p a r a s i t e
and to study f a c t o r s a f f e c t i n g j j i v i t r o c u l t u r e as th e y
p e r t a i n to m e ta b o lic a c t i v i t i e s .
The tapeworm, T aenia
t a e n i a e f o r m i s was ujked i n t h e s e s t u d i e s .
MATERIALS AND METHODS
T ae n ia t a e n i a e f o r m i s i s t h e b ro ad -n eck ed tapeworm,
commonly p a r a s i t i c i n th e d o m estic c a t .
Because o f t h i s
a n a to m ic a l f e a t u r e , R udolphi (1810) named i t T. c r a s s i c o l l i s .
I n 1905, S t i l e s and S teven son d is c u s s e d th e p r i o r i t y o f
names f o r t h i s and o t h e r worms.
They found t h a t B etsch
had named t h i s s p e c i e s , Hvdatijjjgenfl t a e n i a e f o r m i s i n 1786.
W olffhuegel (1911) f i r s t made th e co m b in a tio n , T. t a e n i a e f o r m i s .
( S t i l e s end H a s s a l l , 1 912).
I t I s p ro b a b le t h a t R u d o lp h if s
name f o r t h i s tapeworm e n jo y s more g e n e r a l usag e even to d a y
-2 5 -
th a n does t h e v a l i d name.
Loveland (1894) r e p o r t e d on th e known h o s t s f o r
t h i s worm.
Most o f them were members o f t h e c a t f a m ily .
I t has a l s o been found i n t h e erm in e.
One ca se o f what i s
p ro b a b ly t h i s p a r a s i t e was r e p o r t e d from a c h i l d i n South
Am erica.
The l a r v a l s ta g e o f X» t a e n i a e f o r m i s i s a s t r o b i l i c e r c u s and i s known by t h e name o f C y s tlc e r c u s f a s c i o l a r l s .
I t commonly o c c u rs on t h e l i v e r s o f t h e Norway r a t , th e
b la c k r a t , t h e house mouse, th e w ater v o l e , t h e f i e l d v o l e ,
end t h e lo n g - e a r e d b a t .
H a s s e ll r e p o r t e d t h e s e l e r v e e from
t h e m u s k ra t,a n d Hawkins (1942) r e p o r t e d them from t h e c o t t o n
ra t.
T h is p a r a s i t e h as been r e p o r te d from E urope, A s ia ,
and N orth and South A m erica.
I t s d i s t r i b u t i o n , alo n g w ith
i t s common h o s t s , i s p ro b a b ly w orld-w id e.
The l e n g t h o f th e a d u l t £*. t a e n i a e f o r m i s has been
v a r i o u s l y r e p o r te d as from 10 to 60 cm.
1 .0 - 1 .7 mm. b ro a d .
The s c o le x i s
There a r e f o u r s u c k e rs , m easur­
in g 0 .3 0 - 0 .5 0 mm. i n d ia m e te r .
Two rows o f hooks a r e
p r e s e n t on t h e r o s t e l l u m w ith th e t o t a l number o f hooks
v a r y in g from 29 to 52, b u t u s u a l l y 17 o r 18 o c c u r i n each
row; t h e l a r g e r hooks m easuring 0 .3 2 - 0 .4 2 mm. i n l e n g t h
and t h e s m a lle r ones 0 .1 8 - 0 .2 8 mm.in l e n g t h . The neck i s
ab o u t t h e same b r e a d th as t h e s c o le x .
The f i r s t segm ents
a r e much w ider t h a n th e y a r e lo n g , whereas t h e l a s t segments
-2 6 -
a r e much lo n g e r t h a n wide.
The segm ents a re d e c id e d ly
c r a s p e d o t e , and from t h i s f e a t u r e , th e name T. s e r r a t a
was s e t up by Goeze.
T his name i s n o t v a l i d .
I t has a l s o
been u se d a s a synonym o f T a e n ia p i s i f o r m i s .
H a ll (1923) l i s t e d t h e eggs o f t h i s s p e c ie s as
b ein g 31 - 37 m iera I n d ia m e te r which a g r e e s w ith o r was
ta k e n from R a i l l i e t (1 8 8 6 ). I n Lehmann's M e d iz in isc h e
A tl a n t e n (1 9 1 4 ), B utyra and Marek a r e r e p o r t e d to have found
, ‘V
m easurem ents to be 21 - 27 micr^f. Most eggs measured by
th e a u th o r were found to l i e between th e s e two e x tre m e s,
t h a t i s 27 - 30 m icra i n d i a m e te r .
Eggs from one l a b o r a t o r y -
i n f e c t e d c a t m easured 2 7 .2 - 2 7 .9 m ic ra , whereas th o s e from
a n o t h e r group o f s i m i l a r l y i n f e c t e d c a t s measured 3 0.6 - 34.0
m ic ra .
P o s s i b l y t h i s i n d i c a t e d two d i f f e r e n t s t r a i n s o f
th is sp e c ie s.
The l a r v a l form , C v s t ic e r c u s f a s c i o l a r l s . d e v e lo p s
i n c y s t s on th e s u r f a c e o f t h e l i v e r .
I t has been r e p o r t e d
a s r e a c h in g a l e n g t h o f 45 - 50 cm a lth o u g h none t h a t l e n g t h
were found i n t h e p r e s e n t s tu d y . Growth seems t o be c o n tin u ­
o u s , and t h e r e f o r e t h e i r l e n g t h would depend i n p a r t on th e
l e n g t h o f tim e i n t h e l i v e r , a lth o u g h when v e ry many p a r a si|*es a r e p r e s e n t , growth te n d s to be r e t a r d e d .
T h is l a r v a
I s s t r o b i l a t e d which i s t h e r e a s o n f o r t h e d e s i g n a t i o n
N S tro b ilo c e rc u s.
A sm a ll c a u d a l b la d d e r o c c u rs a t t h e end o f
t h e s t r o b i l u s , and, a c c o rd in g to Vogel ( 1 8 8 8 ) , th e e x c r e t o r y
-2 7 -
system opens I n t o t h i s b la d d e r th ro u g h a s i n g l e p o r e , th e
f o u r l o n g i t u d i n a l c a n a ls having f u s e d .
I n g e n e r a l , th e
s t r u c t u r e o f t h i s l a r v a i s v e ry s i m i l a r t o t h a t o f t h e a d u l t ,
a lth o u g h th e g e n i t a l o rg a n s have n o t a p p e a re d .
/y,
I n 1844, von S ie b o ld noted t h e r e s e n b la n c e o f
C v s t ic e r c u s f a s c i o l a r l s t o T a e n ia c r a s s l c o l l i s ( - t a e n l a e f o r m l s ) .
He knew t h a t £ . f a s c i o l a r l s d id n o t p o s s e s s s e x o r g a n s , b u t
concluded t h a t i t was t h e same worm as T. c r a s s i c o l l l s .b u t
i n an abnormal h o s t i n which c a s e th e sex o rg a n s d id n o t de­
v e lo p .
i n 1851, von S ie b o ld p u b lis h e d a l e t t e r from
K o e l l i k e r who had been i n Glasglow and E dinburgh and which
s t a t e d t h a t A lla n Thompson, a p r o f e s s o r o f anatomy, had
n o ted th e c l o s e c o n fo rm ity o f t h e e n c y s te d worm i n t h e mouse
w ith th e c a t T a e n ia .
I n 1852-53, von S ie b o ld a r r i v e d a t
t h e c o n c lu s io n t h a t th e c y s t i c e r c u s i s t h e l a r v a l s ta g e o f
T a e n ia c r a s s l c o l l i s . b u t i t was K uch enm eister (1857) who
fe d mice w ith t h e eggs o f t h e a d u l t worm, and l a t e r fe d th e
r e s u l t a n t c y s t s t o c a t s , t o remove a l l q u e s t i o n o f th e r e ­
la tio n sh ip .
i n 1878, L e u c k a rt i n v e s t i g a t e d c e r t a i n p h ase s o f t h e
l i f e c y c le o f t h i s p a r a s i t e and came t o th e c o n c lu s io n t h a t
th e s t r o b i l u s o f £ . f a s c i o l a r l s d e g e n e ra te d a f t e r i n g e s t i o n
by a c a t , t h a t o n ly th e s c o le x and neck p e r s i s t , and t h a t
t h e p r o g l o t t i d s o f th e a d u l t a r i s e anew.
T his d is c o v e r y was
d i s p u t e d , and B a r t e l s (1902) a tte m p te d to show t h a t L e u c k a rt
had n o t proved h i s c o n t e n t i o n . However, i n 1937, Joyeux and
-2 8 -
B aer r e p e a te d th e experim en t and L e u c k a rt» s work was con­
f irm e d .
They found t h a t o n ly t h e p o s t e r i o r p a r t o f t h e
s t r o b i l u s d e g e n e r a t e s , a p o r t i o n between o n e - h a l f and on et h i r d o f th e e n t i r e l e n g t h .
The i n t e r m e d ia t e h o s t i n g e s t s t h e eggs i n f e c a l p o l l u t e d fo o d .
The o nchosphere i s fre e d by th e a c t i o n o f
d i g e s t i v e J u i c e s , end a t t a c h e s i t s e l f to th e i n t e s t i n a l w a ll,
through which i t b o r e s .
When i t re a c h e s a c a p i l l a r y o f t h e
p o r t a l system , i t i s c a r r i e d p a s s i v e l y t o th e l i v e r , and
comes to l i e i n a c a p i l l a r y i n t h i s o rg a n .
B u llo ck and
C u r t i s (1924) r e p o r t e d a la r v a ,w h i c h measured abo ut 15 m icra
i n d ia m e te r , found i n a l i v e r c a p i l l a r y 21 h o u rs a f t e r th e
h o s t was fe d i n f e c t i v e f e c e s .
T h is c a p i l l a r y r u p t u r e s ,
u s u a l l y i n about t h r e e d a y s. About t h e s i x t h day, l a r v a e
can be seen on th e s u r f a c e o f t h e l i v e r where th e y ap p e ar
a s sm a ll b l i s t e r s o r v e s i c l e s .
These a u th o r s d is c u s s e d
t h e r e a c t i o n o f th e r a t l i v e r .
"For the f i r s t week t h e
r e a c t i o n ab out th e l a r v a e i s o f t h e n a t u r e o f an e x u d a tiv e
in fla m m a tio n a s s o c i a t e d w ith d e g e n e r a t i o n or n e c r o s i s o f
t h e l i v e r t i s s u e b o rd e rin g t h e o rg a n is m ."
P ro life ra tio n
o f th e t i s s u e around th e p a r a s i t e u s u a l l y b e g in s about t h e
n i n t h day, but where n e c r o s i s i s more pronounced, i t may
be d e la y e d .
The p r o l i f e r a t i v e a c t i v i t i e s r i s e t o a peak
between th e 1 5 th and th e 2 0 th d ay , and t h e r e a f t e r th e y
g r a d u a lly s u b s id e .
The i n c r e a s e i n th e w hite c e l l count i s
-2 9 -
most marked 10 to 30 days a f t e r f e e d in g .
By th e end of 15 weeks, t h e normal l a r v a may
re a c h a l e n g t h o f f i v e t o s i x c e n t i m e t e r s , w ith w e l l pronounced se g m en tatio n and a c a u d a l v e s i c l e .
In fe c tiv e
l a r v a e have been se c u re d i n s l i g h t l y more th a n seven m onths,
a s r e p o r t e d by B u llo c k , C u r t i s , and Dunning ( u n p u b lis h e d ) .
When th e d e f i n i t i v e h o s t e a t s an i n f e c t e d ro d e n t
l i v e r , th e l a r v a e a r e f r e e d i n th e i n t e s t i n e by th e a c t i o n
o f d i g e s t i o n and u s u a l l y come to l i e a l i t t l e a n t e r i o r t o
t h e m iddle o f th e sm all i n t e s t i n e .
As m entioned abo ve, th e
p o s t e r i o r p a r t o f th e s t r o b i l u s t o g e t h e r w ith th e ca u d al
v e s i c l e d e g e n e r a te s and i s l o s t .
A ccording to Joyeux and
Baer (1937J t h i s phenomenon o c c u rs i n about 20 h o u rs , and
a t ab o u t t h e m iddle o f th e rem a in in g s t r o b i l u s , embryonic
a r e a s b e g in to make t h e i r ap p e aran c e n e a r t h e m iddle o f th e
p ro g lo ttid s .
t o 42 d a y s .
Eggs b e g in t o ap p e ar i n th e c a t f e c e s I n 35
Thus t h e com plete l i f e c y c le o f t h i s worm would
r e q u i r e a minimum o f about 250 d a y s .
T h is p a r a s i t e was chosen f o r s tu d y because i t
p r e s e n t s a number of a d v a n ta g e s .
I n th e f i r s t p l a c e , i t s
h o s t s a r e well-known l a b o r a to r y a n im a ls , easy to o b t a i n and
t o keep h e a lth y i n t h e l a b o r a t o r y .
Because o f t h i s f a c t ,
th ey have been s tu d ie d from numerous a n g l e s , and t h e r e i s
c o n s i d e r a b l e knowledge a v a i l a b l e c o n c ern in g th e anatomy and
p h y sio lo g y o f t h e i r o rg s n sy stem s.
Thus a b a s i s i s p ro v id e d
-3 0 -
f o r t h e developm ent o f an a r t i f i c i a l environm ent f o r t h e i r
p a ra s ite s .
F u rth e rm o re , t h e l i f e c y c le o f t h e p a r a s i t e i s
well-know n and t h e h o s t s a r e easy t o i n f e c t .
C ats which may
l o s e t h e i r worms a c c i d e n t a l l y o r u n d e r t h e a c t i o n o f a
verm ifug e can he r e a d i l y r e i n f e c t e d .
On t h e o t h e r hand, th e
f a c t t h a t t h i s worm i n i t s l a r v a l s ta g e i s a t i s s u e p a r a s i t e
i n r a t s p r o v id e s an immunity a g a i n s t c o n tin u e d i n f e s t a t i o n
( M i l l e r and c o -w o rk e rs , 1 9 3 0 - 1 9 3 5 ).
T h e r e f o r e , the i n v e s t i ­
g a to r u s in g such m a t e r i a l knows t h a t th e p a r a s i t e s a r e a l l
o f t h e same age and same f e e d in g .
A nother f a c t o r o f g r e a t im p o rtan ce i s t h a t u s u a l l y
th e p a r a s i t e s from th e r a t l i v e r a r e s t e r i l e .
B u llo c k and
C u r t i s (1924) found two c a s e s o f s ta p h y lo c o c c i i n t e s t i n g
l i v e r fra g m e n ts o f 20 i n f e c t e d r a t s .
I n most c a s e s , t h e r e ­
f o r e , t h e absence o f b a c t e r i a p r o v id e s a ready-m ade c o n t r o l
fe a tu re .
From t h e s ta n d p o in t o f t h e worm, th e absence o f
a gut means t h a t n u t r i t i o n i s d eterm in ed by p e r m e a b i l i t y .
Thus a d e f i n i t e l i m i t i s s e t on th e co m p lex ity o f n u t r i t i v e
m a te ria l.
The e n d -p ro d u c ts o f c a rb o h y d ra te and p o s s i b l y o f
p r o t e i n d i g e s t i o n , t h e sim ple s u g a rs and amino a c id s r e s p e c ­
t i v e l y , seem t o be i n d i c a t e d a s b a s i c f o r tapeworm n u t r i t i o n .
A nother f e a t u r e t h a t I s Im p o rta n t i s t h a t t h e p a r a s i t e , i n
b o th l a r v a l and a d u l t s t a g e s i s r o t u s t and o f a s i z e s u f ­
f i c i e n t f o r s a t i s f a c t o r y l a b o r a t o r y m a n ip u la tio n .
T h is i s
n e c e s s a ry s i n c e t h e worms may have t o be handled a t f r e q u e n t
-3 1 -
i n t e r v a l s i n washing them and t r a n s f e r r i n g them to f r e s h
m edia.
L a b o ra to ry r a t s were fe d eggs c o n t a i n i n g i n f e c t i v e
o n ch o sp h eres by h o ld in g th e mouth open and p l a c i n g th e ma­
t e r i a l f a r back I n t h e t h r o a t w ith a lo n g m ed icin e d ro p p e r.
T h is method was u sed by B u llo c k and C u r t i s (1 9 2 4 ).
The
number o f o n ch o sp h eres p e r drop o f s u s p e n s io n was d e t e r ­
mined, a s e x c e s s iv e i n f e c t i o n s show h ig h e r m o r t a l i t y o f
t h e h o s t , o r , i f t h e anim al s u r v iv e d , l a r v a e i n "crowded"
i n f e c t i o n s tended to be s m a l l e r .
Some r a t s were fe d by
k ee p in g them w ith o u t food f o r 24 h o u rs and th e n g iv in g them
p ie c e s o f b read w ell m oistened w ith i n f e c t i v e onchosphere
s u s p e n s io n .
L a b o ra to ry c a t s were i n f e c t e d by d i s t r i b u t i n g
c y s t s from t h e r a t l i v e r i n chopped meat o r dog fo o d .
I n b e g in n in g a stu d y o f t h e p h y sio lo g y o f t h i s
tapeworm, i t was b e lie v e d n e c e s s a ry to d e te rm in e how lo n g
th e a n im a ls would s u r v iv e o u t s i d e t h e tody o f th e h o s t . V a r i­
ous sim p le media were used i n t h e b e g in n in g and when th e s e
were found to be i n s u f f i c i e n t , more complex ones were t r i e d .
Two methods o f k i l l i n g r a t s were used to o b t a i n
th e l a r v a e o f T a e n ia t a e n i a e f o r m i s . At f i r s t , t h e s e anim als
were k i l l e d by e t h e r a d m i n i s t r a t i o n , b u t i t was th o u g h t t h a t
p o s s i b l y & her might have some e f f e c t on th e e n c y s te d worms.
K i l l i n g was them accom plished by means o f a sh a rp blow on th e
back o f t h e h ead .
D eath r e s u l t s Im m ed iately .
L ittle
-3 2 -
d i f f e r e n c e wes n o te d i n v i t a l i t y o f th e p a r a s i t e s .
L a te r
e t h e r was a g a in u s e d , b u t o n ly to a n a e s t h e t i z e t h e h o s t f o r a s e p t i c rem oval o f th e worms.
In a tte m p tin g t o p r e v e n t th e I n g r e s s of b a c t e r i a ,
s t e r i l e p r e c a u t i o n s were o b s e rv e d .
A fte r k i l l i n g th e ro d e n t,
t h e abdomen o f th e r a t was soaked w ith a l c o h o l , i n c i s i o n was
made w ith s t e r i l e in s tr u m e n ts snd lo b e s o f th e l i v e r w ith a t ­
tach e d c y s t s were removed and p la c e d u n d e r a u to c la v e d mammalian
R ing er s o l u t i o n i n s t e r i l i z e d f i n g e r bow ls.
The te m p e ra tu re
was m a in ta in e d as c l o s e to 3 7 .5 ° C. as p o s s i b l e .
L arvae
were removed from th e c y s t s , o r I n a few e s s e s t h e c y s t i t ­
s e l f was removed from th e l i v e r , below th e s u r f a c e o f t h e
s t e r i l e R in g er s o l u t i o n .
These c y s t s and l a r v a e were q u ic k ly
tra n s f e rre d t o a s i m i l a r c o n t a i n e r t o r i n s e them a s much as
p o s s i b l e from any b lo o d .
They were th e n t r a n s f e r r e d t o th e
r e s p e c t i v e media p r e v io u s ly s t e r i l i z e d and m a in ta in e d a t a
te m p e ra tu r e o f 3 7 .5 ° C.
Types o f c o n t a i n e r s u sed were 50 m l.
and 250 ml. E rlenm eyer f l a s k s and s i x in c h Pyrex t e s t t u b e s .
B a c t e r i o l o g i c a l p ro c e d u re was fo llo w e d i n t r a n s f e r r i n g worms
from one f l a s k t o a n o t h e r , a lth o u g h g r e a t c a r e had t o be ex­
e r c i s e d t o p r e v e n t th e worms from coming i n t o c o n t a c t w ith
t h e flam ed neck o f th e f l a s k .
C o tto n p lu g s were u sed to c l o s e
th e c o n t a i n e r s I n a number o f I n s t a n c e s , b u t u s u a l l y s t e r i l ­
iz e d ru b b e r s to p p e r s were s u b s t i t u t e d .
These s to p p e r s were
t r e a t e d a f t e r t h e method o f Buchsbaum (1936) and were a u t o -
-3 3 -
c la v e d i n d i v i d u a l l y wrapped I n c h e e se c l o t h , I n a b e a k e r
cov ered w ith many l a y e r s o f c h e ese c l o t h .
A w ire g r a t e was
p ie c e d i n th e bottom o f t h e b e a k e r so t h a t condensed w ater
would n o t be soaked up by t h e wrapped s t o p p e r s .
A number o f media were u sed i n p r e l i m i n a r y e x p e r i ­
m en ts.
F i n a l l y , Tyrode s o l u t i o n was d e c id e d upon a s t h e
base f o r most o f th e c u l t u r e e x p e rim e n ts , a lth o u g h o c c a s io n ­
a l l y mammalian R in g er s o l u t i o n was s u b s t i t u t e d .
Mammalian
o rg a n s g iv e much b e t t e r r e a c t i o n s and m a in ta in m o t i l i t y l o n g e r
i n Tyrode s o l u t i o n which c o n ta i n s magnesium th a n i n o r d in a r y
R ing er-L ock e s o l u t i o n s which u s u a l l y do n o t c o n t a i n t h i s
s a l t , ( M i t c h e l l , 1 9 3 8 ).
f e r and g lu c o s e .
Tyrode s o l u t i o n a l s o c o n t a i n s b u f­
I t cannot be a u to c la v e d i n th e f i n a l form,
b u t t h e sodium b ic a r b o n a te can be a u to c la v e d a s a se p a ra te
s o l u t i o n and th e n added t o th e o t h e r a u to c la v e d components
la te r.
Sometimes disodium p h o sp h a te was s u b s t i t u t e d f o r t h e
sodium b i c a r b o n a t e , and v a r io u s In o r g a n ic n i t r o g e n so u rc e s
were added i n c e r t a i n e x p e rim e n ts .
i n th e p r e p a r a t i o n o f more complex m edia, D Ifco*s
t r y p t o n e was u sed whole, but i n most c a s e s D ifc o * s pep tone
and t r y p t o n e were d i g e s t e d w ith P a n c r e a t i n fo r a number o f
h o u r s , and t h i s d i g e s t was used f o r a so u rce of amino a c id s
i n some o f th e m edia.
I n some c a s e s , d i g e s t s o f r a t and c a t
t i s s u e s were u se d as amino a c id s o u rc e s i n a tte m p ts t o f in d
e s s e n t i a l amino a c id s o u r c e s .
-3 4 -
The d i g e s t s p re p a re d from th e D ifco p r o d u c ts were
u t i l i z e d a s l i q u i d s i n some c a s e s , b u t t h e u s u a l method was
to p re p a re t h e s e am in o -ac id complexes i n a g a r , th e n th e
worms were p la c e d i n Tyrode o r i t s m o d i f i c a t i o n s on to p o f
th e a g a r , so t h a t a g ra d u a l seepage o f amino a c id s from th e
ag 8 r would o c c u r.
I n c e r t a i n e x p e rim e n ts , a c r i f l a v i n e
(N a tio n a l A n i l in e and Chemical Co.) and th e b i l e s a l t ,
sodium t a u r o c h o l a t e , were added to t h e amino a c i d - a g a r com­
p le x .
U s u a lly th e worms were t r a n s f e r r e d to f r e s h media
a t f r e q u e n t i n t e r v a l s , but i n some c a s e s th e medium was un­
changed d u rin g th e co u rse o f t h e e x p e rim e n t.
A c t i v i t y was t h e p r i n c i p a l c r i t e r i o n f o r d e t e r ­
mining th e l e n g t h o f l i f e o f worms l i v i n g i n a r t i f i c i a l media.
A number o f a tte m p ts were made t o u t i l i z e t h e c u r r e n t from
an i n d u c t i o n c o i l (H arvard A pparatus Company) to s t i m u l a t e
th e worm t o movement,but t h i s d id not prove s a t i s f a c t o r y and
i t was th o u g h t t h a t some i n j u r y m ight a c c ru e to th e p a r a ­
s i t e s , so m ild shaking of th e c o n t a i n e r and o c c a s i o n a l g e n t l e
p ro b in g were u t i l i z e d .
The shak ing o f t h e c o n t a i n e r u s u a l l y
p ro v id ed s u f f i c i e n t s t i m u l a t i o n , p ro b a b ly b o th m e c h a n ic a lly
and c h e m ic a lly , so t h a t t h e worms moved, i f th e y w ereable to
do so .
I t i s p o s s i b l e t h a t t h e r a t e o f oxygen consum ption may
p ro v id e a method f o r i n d i c a t i n g v i t a l i t y , a lth o u g h In e x tre m e ly
sm all amounts o f oxygen, th e p o s s i b i l i t y o f e r r o r i s so l a r g e ,
th a t I t i s d o u b tf u l w hether t h e r e s u l t s would be sound s t a t i s ­
tic a lly .
The W inkler method was u se d f o r measurements o f
oxygen consum ption.
A number o f m o d i f i c a ti o n s f o r m easur­
in g oxygen i n sm all amounts o f f l u i d have been r e p o r t e d I n
th e l i t e r a t u r e .
Fox and W in g field (1939) d i s c u s s e d an ap­
p a r a t u s s u i t a b l e f o r such d e t e r m i n a t i o n s .
I n th e p r e s e n t
s tu d y , a 30 ml. hypodermic s y r in g e was u sed f o r a r e s p i r a t o r y
chamber.
A f t e r f i l l i n g i t w ith t h e d e s i r e d amount o f f l u i d
and p l a c i n g th e worms i n I t , th e s y rin g e was i n v e r t e d and any
bubble o f a i r p r e s e n t was fo rc e d o u t .
A n e e d le whose s h a f t
was c lo s e d w ith a ru b b e r s to p p e r was u sed to c l o s e t h e neck
o f th e s y r in g e .
A 10 ml. hypodermic s y r in g e was used f o r a
r e a c t i o n chamber.
For t r a n s f e r r i n g f l u i d from one chamber
to th e o t h e r , a n e e d le w ith a p ie c e o f ru b b e r s to p p e r s e t
midway on t h e s h a f t was p la c e d on t h e i n v e r t e d r e s p i r a t o r y
sy rin g e .
The p lu n g e r o f th e r e a c t i o n chamber was c o m p le te ly
down a t t h i s tim e , and t h e neck o f t h e r e a c t i o n chamber was
p la c e d o v er t h e open end o f th e n e e d le on th e r e s p i r a t o r y
chamber and h e ld f ir m ly a g a i n s t th e p ie c e o f ru b b e r s to p p e r
on th e s h a f t of t h i s n e e d le so t h a t a t i g h t c o n n e c tio n was
ac co m p lish ed .
L iq u id was f o r c e d i n t o f i l l t h e b l in d space
i n t h e neck of t h e r e a c t i o n s y rin g e and t h e a i r h e re was
fo rc e d o u t .
The amount o f f l u i d to be t e s t e d was th e n fo rc e d
i n t o t h e r e a c t i o n chamber and t h i s was u s u a l l y accom plished
-5 6 -
w ith no b ubb le o f a i r .
When such a bubble d id o c c u r however,
th e r e a c t i o n s y r in g e was im m ed iately i n v e r t e d and t h e bu bble
e je c te d .
T^reupon, t h e n e c e s s a r y amount o f a l k a l i n e - i o d i d e
s o l u t i o n and manganese c h l o r i d e s o l u t i o n were drawn i n t o th e
r e a c t i o n chamber and allow ed t o r e a c t f o r two m in u te s w ith
i n t e r m i t t e n t s h a k in g .
T h is chamber was k e p t a i r t i g h t by
means o f a hypodermic n e e d le base which had been s e a le d s h u t .
When t h e r e a c t i o n was c o m p lete, c o n c e n tr a te d h y d r o c h lo r ic
a c id was drawn i n t o t h e r e a c t i o n chamber.
T h is r e l e a s e d th e
i o d in e and th e c l e a r y e llo w s o l u t i o n was p la c e d in a sm all
erlen m ey er f l a s k and t i t r a t e d w ith 0 .0 0 1 sodium t h i o s u l p h a t e
w ith s t a r c h a s an i n d i c a t o r .
The t h i o s u l p h a t e was s ta n d a r d ­
iz e d a g a i n s t i o d a t e a c c o rd in g to th e d i r e c t i o n s o f P e t e r s
and Van S ly k e , (1952)•
Two c u b ic c e n tim e t e r s o f s o l u t i o n
were found s a t i s f a c t o r y f o r measurement o f oxygen a t each
d e t e r m i n a t i o n , and a s e r i e s o f d e te r m i n a t i o n s o f oxygen con­
sum ption were made, b o th on worms i n normal mammalian R inger
s o l u t i o n and th o s e i n v a r io u s c o n c e n t r a t i o n s o f p o ta ssiu m
cyan id e i n R in g e rs .
T h is drug was used s in c e i t i s a s p e c i ­
f i c p o iso n f o r th e W arburg-K eilen cytochrom e system o f
c e ll u l a r o x id a tio n .
S tu d ie s on th e r e d u c t io n o f m ethylene b lu e were
made w ith s ta n d a r d Thunberg t u b e s , e v a c u a te d o f a i r .
A
com parison was made betw een m ethylene b lu e r e d u c t i o n in
mammalian R inger s o l u t i o n and i n mammalian R in g e r w ith one
p e r c e n t g lu c o s e s u b s t r a t e .
Measurements o f th e change i n
c o l o r o f t h e m ethylene b lu e were made e l e c t r o m e t r i c a l l y .
A n aerob ic s t u d i e s were made by v a r io u s m ethods.
One o f t h e s e i s a s ta n d a r d b a c t e r i o l o g i c a l te c h n iq u e i n
which f r e s h l y ground meat i s p la c e d i n t h e lo w er h a l f o f
s ta n d a r d s i x in c h t e s t tu b e s and covered w ith Tyrode
mammalian R in g e r.
An a n a e ro b ic environm ent e x i s t e d a t t h e
bottom o f such tu b e s .
Another method was u t i l i z a t i o n o f a
sodium t h i o g l y c o l l a t e medium c o n ta in in g p o rk i n f u s i o n s o l i d s ,
p e p to n e , and d e x t r o s e , (Brewer, 1 9 4 0 ).
M ethylene b lu e i s
p r e s e n t i n t h i s l i q u i d medium a s en i n d i c a t o r .
T h is medium
was u sed i n s i x i n c h t e s t tu b e s o f sm all d ia m e te r .
The
s o l u t i o n i s a e r o b i c a t th e to p a s i n d i c a t e d by th e m ethylene
b lu e and becomes s u c c e s s i v e l y more a n a e ro b ic toward th e
b o tto m .
C o n seq u en tly t h e h e ig h t o f th e column o f medium
can be a d e te rm in in g f a c t o r i n t h e amount o f a n a e r o b i o s i s
a t t h e bottom o f th e tu b e .
I n some t e s t s , an a n a e ro b ic
c o n d i t i o n was approached by p l a c i n g th e worms i n t e s t tu b e s
i n th e medium and c o v e rin g th e s o l u t i o n w ith a t h i c k l a y e r
o f N u jo l.
Then a llo w in g o n ly a minimum o f a i r space f o r
d is p la c e m e n t, t h e t e s t tu b e was t i g h t l y c lo s e d w ith a r u b ­
ber sto p p e r.
A f t e r t h e l a r v a e have u t i l i z e d th e oxygen a l ­
re ad y p r e s e n t i n t h e medium, th e y w i l l have to r e s p i r e a n ­
a e r o b ic a lly to su rv iv e .
Cat serum was o b ta in e d from an a n a e s t h e t i z e d c a t
by means o f a s t e r i l e c i t r a t e d s y r in g e and th e blood was
drawn d i r e c t l y from t h e h e a r t .
A f t e r t h e blood had coagu­
l a t e d i n s t e r i l e v e s s e l s , t h e serum was d ec an ted and p la c e d
i n r u b b e r - s to p p e r e d s t e r i l e am poules.
As f u r t h e r p r e c i p i ­
t a t i o n sometimes o c c u r r e d , o n ly th e serum i n t h e u p p e r
p a r t o f th e ampoule was used and t h i s was drawn o f f d i r e c t l y
by hypoderm ic s y r in g e .
Chick embryonic e x t r a c t was made from eggs o f t e n
and tw e lv e days in c u b a t i o n .
The embryonic m a t e r i a l was
a s e p t i c a l l y removed and c u t i n t o m inute fra g m e n ts w ith
s te r ile sc isso rs.
T h is m a t e r i a l was th e n p la c e d i n f l a s k s
w ith e q u a l amounts o f F in g e r s o l u t i o n .
Only t h e su p e rn a ­
t a n t f l u i d was used f o r e x p e rim e n ts.
S u rfa c e t e n s i o n was reduced by t h e u s e o f b i l e
s a l t s and o f h e x y l r e s o r c i n a l (S . T .3 7 ) .
M easurements o f
pH were made in th e LaMotte Com parator f o r t h e most p a r t ,
b u t , i n some l a t e r e x p e rim e n ts , pH p a p e rs (P a u l Prank) were
u tiliz e d .
E ecause o f th e l o c a t i o n o f th e l a r v a e o f T ae n ia
t a e n i a e f o r m i s i n th e l i v e r , i t was c o n s id e r e d t h a t u r i c
a c i d , a l l a n t o i n , and even u r e a might have some e f f e c t on t h e
m etabolism o f th e s e worms. Robinson and W ilson (1 9 3 9 a,b )
showed t h e r e l a t i o n s h i p o f c o n c e n t r a t i o n o f t h e enzyme u r e a s e
w ith th e r e o r g a n i z a t i o n o c c u r r i n g I n th e pupa o f Phormia
re g in a .
U rease i s s p e c i f i c f o r u r e a and t h e c o n c e n t r a t i o n
-3 9 -
was g r e a t e s t between 50 and 80 hours o f p u p a l l i f e , f a l l ­
in g s h a r p l y a t th e end o f th e p u p al p e r i o d .
T h is i n d i c a t e s
t h a t u r e a may be im p o rta n t i n t h i s r e o r g a n i z a t i o n p r o c e s s .
Matthews (1956) has p o in te d o u t t h a t u r e a , which p e n e t r a t e s
c e l l s r e a d i l y , has powers o f d i s s o l v i n g p r o t e i n s , s t a r c h e s ,
and o t h e r s u b s ta n c e s .
on g ly co g en .
T h e r e fo re i t could c o n c e iv a b ly a c t
He a l s o p o in te d o u t t h a t a l l a n t o i n may be t h e
c u r a t i v e p r i n c i p l e i n t h e h e a li n g o f c e r t a i n wounds.
RESULTS
PART I .
ATTEMPTS AT IN VITRO CULTURE
1. C u ltu r e e x p e rim e n ts u t i l i z i n g media w ith o u t aminon itro g e n sources.
On A p r il 2 5 ,1 9 3 8 , s i x l a r v a e o f T a e n ia t a e n i a e ­
fo rm is from a f r e s h l y k i l l e d r a t were p la c e d i n each o f two
fla sk s.
One c o n ta in e d n o n - s t e r i l e Tyrode s o l u t i o n ; t h e
o t h e r c o n ta in e d an e q u i v a l e n t amount o f th e same Tyrode s o l u ­
t i o n (s to c k ) to which p heno l was added t o make a 0 .0 1 p e r
cent s o lu tio n of c a rb o lic a c id .
Worms i n each o f th e s e
c o n t a i n e r s were a l i v e May 1 2 th , and t h e r e f o r e l i v e d
mum of 13 - 14 d a y s .
bljA
m in i­
The media were changed seven tim e s
d u rin g t h i s p e r i o d .
Tyrode s o l u t i o n u sed i n two e x p e rim e n ts i n th e
F a l l o f 1938, m a in ta in e d one group o f worms a l i v e 156 h o u rs
and a n o th e r group a p p ro x im a te ly 50 h o u rs , when t h e in c u b a ­
t o r o v e r - h e a te d and k i l l e d th e worms.
-4 0 -
A s e r i e s o f e x p e rim e n ts was begun December 2,1938
u s in g Tyrode s o l u t i o n .
One gram o f sodium b ic a r b o n a te i n ­
s te a d o f t h e custom ary 0 .5 gm. was u sed i n t h i s Tyrode s o lu ­
tio n .
The i n i t i a l pH o f t h i s medium a f t e r a u t o c la v in g was
w e ll above 9 . 0 , and h y d r o c h lo r ic a c id was added t o b rin g i t
to 8 . 4 - 9 . 0 .
T a b le I g iv e s th e pH r e a d in g s o f t h e media f o r
two groups o f worms.
The av erag e e la p s e d tim e between suc­
c e s s i v e pH re a d in g s was a l i t t l e more t h a n 15 h o u rs .
The
a v e ra g e pH drop was 0 .2 5 f o r th e media i n th e c o n t a i n e r w ith
one worm.
The a v e ra g e d rop w ith t h r e e worms was 1 .3 4 pH.
T h is change i n th e pH o f t h e s o l u t i o n c o n ta in in g t h r e e worms
was more th a n t h r e e tim e s a s g r e a t a s t h e c o rre s p o n d in g change
w ith th e s i n g l e worm and may be due i n p a r t to t h e f a c t t h a t
movement o f one worm p ro b a b ly s t i m u l a t e s o t h e r s p r e s e n t i n
th e same f l a s k to move and th e g r e a t e r a c t i v i t y would p ro ­
duce g r e a t e r p h y s i o l o g i c a l changes.
e r s were changed once o r tw ic e a d a y .
Media i n t h e s e c o n t a i n ­
There was no ev id en c e
o f b a c t e r i a l development u n t i l December 1 6 th , when C o n ta in e r I I ,
w ith th e t h r e e worms, appeared to have some p r e c i p i t a t e p r e s ­
e n t and a s l i g h t c lo u d in e s s was o b se rv e d .
Two o f th e worms
i n t h i s c o n t a i n e r were dead 25 h o u rs l a t e r and were removed.
The t h i r d worm i n t h i s v e s s e l was s t i l l a l i v e December 1 8 th ,
16 days a f t e r t h e s t a r t o f t h e ex p e rim e n t.
S t e r i l e Tyrode s o l u t i o n m odified by th e s u b s t i t u ­
t i o n o f disodium p h o sp h ate f o r t h e u s u a l b ic a r b o n a te was used
-4 1 -
th ro u g h o u t most o f a s e r i e s begun December 15,1 9 3 8 . T ab le I I
shows t h e pH changes o c c u r r i n g u n d er t h e c o n d i t i o n s o f t h e
e x p e rim e n t.
One o f th e worms i n C o n ta in e r I was much s m a lle r
th a n t h e o t h e r s which were a p p ro x im a te ly e q u a l i n s i z e w ith
th o s e i n th e second c o n t a i n e r .
The av e ra g e f a l l i n ^H f o r
themedium i n C o n ta in e r I I was 1 .3 4 , whereas t h e av erag e f a l l
i n C o n ta in e r I , which had th e sm a ll worm, was on ly 1 . 0 .
a v e ra g e i n t e r v a l between r e a d in g s was 1 6 .1 h o u rs .
worm was dead on December 20 and was removed.
The
The sm all
The medium i n
C o n ta in e r I , i n which two worms re m a in e d , ap peared cloudy
when t h e r e a d in g
on December 22 was ta k e n .
were te r m in a te d th e fo llo w in g day.
The pH r e a d in g s
Four o f t h e re m ain in g f i v e
worms used i n t h i s ex p erim en t were a l i v e 197 h o u rs , and th e
f i f t h , 293 h o u rs (on December 27th) a f t e r th e b eg in n in g o f
t h i s e x p e rim e n t.
On March 1,1939 a r a t was k i l l e d .
There were be­
tween 150 and 200 c y s t s on th e l i v e r o f t h i s a n im a l.
Most o f
t h e c y s t s were ab o u t f i v e m i l l i m e t e r s i n d ia m e te r , and th e
average l e n g t h o f t h e worms removed from t h e i r c y s t s and w e llexpanded was 12 t o 15 mm.
Disodium pho sp h ate re p la c e d t h e
sodium b ic a r b o n a te i n th e Tyrode u sed i n t h i s e x p e rim e n t. A
p o r t i o n o f t h i s s o l u t i o n was u sed f o r th e c o n t r o l medium, and
a 0 .0 1 p e r c e n t s o l u t i o n o f u r e a was made up In an e q u iv a l e n t
amount o f Tyrode.
As a check on th e u r e a - T r y r o d e , a 0 .0 1 p e r
c e n t s o l u t i o n o f ammonium c h l o r i d e was made up i n a n o th e r
-4 2 -
volume o f t h i s Tyrode.
F iv e worms were p la c e d i n each s o l u ­
t i o n and th e y were changed t o f r e s h media each day o f t h e
e x p e rim e n t, which te rm in a te d on March 8 t h .
u re a-T y ro d e w ere s t i l l a l i v e on t h i s day.
worms i n th e
S l i g h t movements
were v i s i b l e and t h e g e n e r a l ap pearance was good.
worms l i v e d 161 h o u rs •
These
L arvae i n th e ammonium c h l o r i d e -
Tyrode were dead and t h e i r ap p e aran c e was abnorm al, whereas
t h e worms i n th e c o n t r o l d i s i n t e g r a t e d upon s l i g h t shaking
o f th e c o n t a i n e r .
The amount of d iso d iu m p h o sp h a te in t h i s
m o d ifie d Tyrode s o l u t i o n was more th a n d o u b le t h a t used p r e ­
v i o u s ly in which worms l i v e d 12 d a y s, b u t s in c e i t was from
t h e same s to c k s o l u t i o n as t h a t made up with th e u r e a , i n which
th e worms were a l i v e a t th e end o f seven d a y s, i t i s d i f f i ­
c u l t t o e x p l a i n why i t seems to be th e l e a s t e f f e c t i v e o f
t h e t h r e e s o l u t i o n s u sed i n t h i s e x p e rim e n t.
On March 1 3 th an a tte m p t was made to c u l t i v a t e th e
l© rvae o f T aen ia t a e n i a e f o r m i s In th e m o d ified Tyrode s o l u ­
t i o n c o n t a i n i n g 0 .0 1 p e r c e n t u r e a .
On t h i s d a t e , fo u r worms
from f o u r d i f f e r e n t r a t h o s t s were p la c e d t o g e t h e r i n an
E rlenm eyer f l a s k .
c l a v in g was 8 . 4 .
The i n i t i a l pH o f t h i s medium a f t e r a u t o On March 1 6 th , t h e worms were s e p a r a te d
i n t o two groups o f two ea ch .
The r e s u l t s o f t h i s ex perim ent
a r e t a b u l a t e d in T ab le I I I .
The worms l i v e d a p p ro x im a te ly e i g h t days (192 h ours)
in th i s so lu tio n .
Because o f c o n ta m in a tio n I n t r a n s f e r , t h i s
-4 3 -
medlum was n o t b a c t e r i a - f r e e , and s e v e r a l d ro p s o f o l d
medium d r a in e d d i r e c t l y from th e worm and p la c e d i n a f l a s k
o f f r e s h medium produced a pH drop o f 1 .5 i n 1 8 .5 h o u rs .
T h e r e f o r e , a c e r t a i n p e rc e n ta g e o f t h e pH change i n t h i s
s e r i e s was due to t h e p re se n c e o f b a c t e r i a , and v a lu e o f
t h e d a t a c o n s e q u e n tly I s l i m i t e d .
The average change in pH
i n t h i s s e r i e s b e fo re th e worms were s e p a r a te d i n t o two
groups was 2 .9 f o r an av erag e p e r io d o f 9 .4 h o u r s .
A f te r
s e p a r a t i o n , t h e av erag e drop was 3 .0 f o r Group I composed o f
two worms, w hile f o r Group I I t h e d ro p was 2 .9 5 pH d u rin g
an av e ra g e p e r io d o f 1 0 .5 h o u r s .
As l a t e r work te n d s t o show,
th e r e s p i r a t i o n r a t e , when th e p a r a s i t e has f i r s t been p la c e d
i n an a r t i f i c i a l medium and a f t e r th e i n i t i a l shock h a s worn
o f f , i s somewhat more r a p id th a n d u rin g l a t e r p e r i o d s .
T h e r e f o r e , i t seems s u r p r i s i n g t h a t t h e pH d ro p , i f t h a t i n ­
d i c a t e s r e s p i r a t o r y a c t i v i t i e s o f th e worms i n any s e n s e ,
shou ld rem ain a t th e same l e v e l w hile th e worms a r e g e t t i n g
" o l d e r " , n o t to m ention th e f a c t t h a t t h e i r numbers were
h a lv e d .
S in ce th e worms were dead on th e e i g h t h day, th e
p ro b a b le e x p la n a tio n i s t h a t , e v e n b e f o r e t h i s d a t e , c e r t a i n
p o r t i o n s o f th e worms were b eg in n in g t o decompose w ith th e
r e s u l t t h a t a h e a v ie r c o n ta m in a tio n o f b a c t e r i a was c a r r i e d
i n t o th e f r e s h medium a t tim e o f changing.
T his decomposi­
t i o n a l s o would i n c r e a s e b a c t e r i a l food s o u r c e s .
I t should
be p o in te d o u t , however, t h a t t h e pH change o f 1 .5 caused
-4 4 -
by t h e b a c t e r i a was ta k e n toward th e end o f t h e s e r i e s when
th ey sh o u ld be a t maximum and a l s o t h a t t h a t change r e s u l t e d
a f t e r 1 8 .5 hours a t 3 7 .5 ° c e n t i g r a d e .
The averag e change i n
pH f o r t h e media c o n t a i n i n g t h e worms was 3.7 5 f o r t h i s
same p e r io d o f tim e .
I t seems l i k e l y , t h e r e f o r e , t h a t th e
r e s u l t s i n T ab le I I I i n d i c a t e a p r o g r e s s iv e d e c re a s e i n
r e s p i r a t o r y a c t i v i t i e s o f th e m etazoa coupled w ith a p ro ­
g re s s iv e i n c r e a s e o f such e f f e c t s from b a c t e r i a .
On August 10, f i v e worms from an i n f e c t e d r a t were
p la c e d In 20 c c . o f a u to c la v e d mammalian R in g er s o l u t i o n and
f iv e d ro p s o f h y e x l r e s o r c i n o l (S .T .3 7 ) were added s im u lta n ­
e o u s ly .
S i m i l a r l y , fo u r o t h e r l a r v a e from th e same r a t were
p la c e d i n 20 c c . of a u to c la v e d R in g er s o l u t i o n f o r a c o n t r o l .
The worms were changed t o f r e s h , s t e r i l e media t h r e e tim e s
i n t h e c o u rse o f th e e x p e rim e n t.
Movement o f worms was d i s ­
cerned i n bo th f l a s k s alm ost seven days (161 hours) a f t e r t h e
s t a r t o f t h e ex p e rim e n t.
I n t h e S p rin g o f 1940, in t h e co u rse o f r e s p i r a ­
to r y ex p e rim e n ts on th e l a r v a e o f T aenia t a e n i a e f o r m i s . 36
worms from the same r a t were p lace d i n 150 c c . o f s t e r i l e
mammalian R in g er s o l u t i o n , A p r i l 1 2 th .
These worms were
changed t o f r e s h R in g er A p r i l 2 5 th and m o s t , i f not a l l , w e r e
s t i l l a liv e .
On A p r i l 2 7 th , th e y were a g a in changed to f r e s h
medium and a few d ro p s o f a 20 p e r c e n t g lu c o se s o l u t i o n was
added.
T h is l a t t e r medium was used i n th e su b seq u en t changes
-4 5 -
made on May 3, May 4 , and May 6 .
Some l a r v a e were s t i l l
a l i v e on t h i s l a s t d a t e which was 24 days (576 h o u rs) a f t e r
t h e b e g in n in g o f th e s e r i e s .
T here was no s ig n o f decom­
p o s i t i o n among t h e worms l e f t i n th e f l a s k a t 3 7 .5 ° c e n t i ­
grade upon su b seq u en t e x a m in a tio n s e v e r a l weeks l a t e r .
O th e r worms u t i l i z e d i n v a r io u s r e s p i r a t o r y e x p e r i ­
ments about t h i s same tim e and which were s u b j e c t to con­
s i d e r a b l e h a n d lin g were m a in ta in e d a l i v e f o r th e fo llo w in g
m inim al p e r io d s : seven d ay s, tw e lv e d a y s , and f i v e d a y s .
These l a r v a e were k e p t i n s t e r i l e mammalian R in g er s o l u t i o n
between r e s p i r a t i o n e x p e rim e n ts .
2 . C u ltu r e e x p e rim e n ts u t i l i z i n g media c o n ta in in g
a m in o -n itro g e n s o u r c e s .
I n the Summer and F a l l o f 1937, worms were removed
from r a t s and p la c e d i n 1 .5 p e r c e n t T ry p to n e (D ifco ) made
up i n d i s t i l l e d w a te r.
T h is medium was n o t s t e r i l i z e d .
A f t e r t e n d a y s , a lth o u g h no movement was d i s c e r n i b l e , th e
ap p e aran c e o f th e worms was good.
I n J a n u a ry 1938, one worm
was m a in ta in e d i n a 5 p e r c e n t T ry p to n e s o l u t i o n to which
p h o sp h ate b u f f e r had been added.
s o l u t i o n was 7 . 3 .
The i n i t i a l pH o f t h i s
T h is worm was a l i v e a t t h e end o f s i x d a y s ,
and was l a t e r t r a n s f e r r e d t o Mammalien R inger s o l u t i o n , but
no movement was d i s c e r n i b l e on t h e e i g h t h day.
B a c t e r i a were
p re se n t.
A medium composed o f T ry p to n e , and mammalian R inger
s o l u t i o n and b u ffe re d w ith p h o sp h ate t o a pH o f 7 .3 a f t e r
-4 6 -
a u t o c la v i n g was made and t o t h i s a t r a c e o f sodium t a u r o c h o l a t e
was added.
One l a r v a from each of two r a t s was p la c e d i n
th e medium i n s t e r i l e c o tto n -p lu g g e d E rlenm eyer f l a s k s on
F eb ru a ry 2 ,1 9 3 8 .
T r a n s f e r s were made t o f r e s h media on
F e b ru a ry 7 , 11, 19, and 23.
No movement was d i s c e r n i b l e a f t e r
th e f i f t h d ay , but t h e worms rem ained i n good c o n d itio n f o r
s e v e r a l days t h e r e a f t e r , and t h i s m a t e r i a l was n o t d is c a r d e d
u n t i l one o f th e f l a s k s became co n tam in ated i n t r a n s f e r .
On F e b ru a ry 2 5 th , one worm from each o f two r a t s
was p la c e d in s t e r i l e Erlenm eyer f l a s k s c o n ta in in g t h i s
medium.
T h is ex p e rim en t was te r m in a te d about two days l a t e r
by I n c u b a to r t r o u b l e .
On A p r il 1 8 th , two worms were p la c e d In s e p a r a t e
c o n t a i n e r s u s in g t h e same medium t o which m acerated l i v e r
from an i n f e c t e d r a t was added.
One o f t h e s e worms was a c ­
c i d e n t a l l y k i l l e d by h e a t two days l a t e r , w h ile th e worms
were b ein g p la c e d In f r e s h m edia.
The o t h e r worm l i v e d f i v e
d ay s.
On A p r i l 2 5 th , f i v e worms were p la c e d i n each o f
two f l a s k s .
One o f t h e s e groups was p la c e d i n a medium
formed o f p a n c r e a t i n - d i g e s t e d "im m ature" l a r v a e of T . t a e n i a e ­
fo rm is which had been k i l l e d by h e a tin g to 45° c e n t i g r a d e .
The medium f o r th e second group o f worms was composed o f
e q u a l amounts of t h i s d i g e s t and o f mammalian R inger s o l u ­
tio n .
The f i r s t medium seemed to be t o x i c Im m ed iately , b u t
-4 7 -
t h e worms i n th e second medium l i v e d a minimum of 48 h o u rs .
On A p r i l 1 8 ,1 9 3 9 , a number of young l a r v a e were
p la c e d In each o f th e fo llo w in g s o l u t i o n s : 20 c c . o f Tyrode
s o l u t i o n o v e r a p ep to n e (D ifco ) d i g e s t made up i n two p e r
cent ag ar;
and 20 c c . of m o d ified (diso dium p h o sp h ate)
Tyrode s o l u t i o n ov er an i n f e c t e d r a t - l i v e r d i g e s t made up
i n t h r e e p e r c e n t a g a r to which two p e r c e n t g e l a t i n had
been added.
The worms l i v e d a p p ro x im a te ly f i v e days in th e s e
m edia, which were changed fo u r tim e s d u rin g t h i s i n t e r v a l .
On May 7 th , an experim ent was s e t up w ith nona u to c la v e d m edia.
Twelve o r more l a r g e worms were added to
each o f t h r e e f l a s k s : one o f which c o n ta in e d 20 cc. Tyrode
s o l u t i o n ; a second c o n ta in e d an e q u i v a l e n t of t h i s seme s to c k
Tyrode o v er p l a i n a g a r (two p e r c e n t ) ; and th e t h i r d c o n ta in e d
a n o t h e r volume o f th e Tyrode o v e r a pep tone d i g e s t made up
i n two per cent a g a r .
o f th e f l a s k s .
One drop o f t o l u o l was added to ea ch
No movement was d i s c e r n i b l e even on th e second
day and when i t was e s t a b l i s h e d t h a t zhe worms were dead,
t h e experim ent was te r m i n a t e d .
On May 1 0 th , a n o th e r s e r i e s was s e t u p , u s in g Tyrode
o v e r p l a i n a g a r and o v e r p e p to n e - d ig e s t a g a r .
The i n i t i a l
pH o f t h e Tyrode a f t e r a u t o c la v in g was 8 .6 and t h e worms were
t r a n s f e r r e d to f r e s h media s ix tim e s .
A pH r e a d in g was
ta k e n each tim e im m ed iately b e fo re ch an g in g t h e medium. The
a v e ra g e pH drop fo r t h e s o l u t i o n o v er p l a i n s g a r was 1 .5 , w hile
-4 8 -
i t was 1 .9 f o r t h e s o l u t i o n w ith th e p e p t o n e - d i g e s t a g a r
f o r tim e p e r io d s which averaged a p p ro x im a te ly 24 h o u rs .
T h is low er pH re a d in g i n th e l a t t e r c a s e may he acco u n ted
f o r by th e p re s e n c e o f amino a c i d s .
The worms were a l i v e
s l i g h t l y more th a n s ix days (146 h o u rs) in each c a s e .
May 1 5 th , t h e p e p to n e - d ig e s t a g a r was a g a in t r i e d .
On
The
a v e ra g e pH r e a d in g a f t e r tim e p e r io d s s i m i l a r to th o s e o f
t h e May 10th s e r i e s was 6 .7 5 , so t h a t a drop o f 1 .8 5 i n pH
had o c c u r r e d .
The worms were a l i v e in t h i s s o l u t i o n a p p r o x i­
m ately s ix d a y s.
On May 2 0 th , t h r e e worms were removed from a r a t
l i v e r and p la c e d i n 20 cc. o f Tyrode s o l u t i o n o v er th e t r y p to n e d i g e s t i n one p e r c e n t a g a r t o which a sm all amount
o f sodium t a u r o c h o l a t e had been added.
These worms were
a c t i v e a t 44 h ours when they were changed to f r e s h medium
f o r th e second t im e .
T h ir ty - tw o hou rs l a t e r , th e y were not
o n ly d ea d , b u t had d i s i n t e g r a t e d .
In a r e p e t i t i o n o f t h i s
ex p e rim en t on Ju ne 7 t h , two l a r v a e were m ain tain ed a l i v e
betw een fo u r and f i v e d ay s.
Another r e p e t i t i o n begun on
J u n e 1 6 th , m a in ta in e d a s i n g l e l a r v a f o r o v er seven d a y s .
T h is medium showed c o n ta m in a tio n , but f i v e changes to f r e s h ,
s t e r i l e medium helped t o keep th e b a c t e r i a l growth i n check.
I n two subsequent e x p e rim e n ts on Ju n e 1 9 th and Ju n e 2 9 th ,
la rv a e of
ta e n l a e f o r m l s were k e p t a l i v e i n t h i s same
medium f i v e and n in e days r e s p e c t i v e l y .
The form er medium
-4 9 -
was s t e r i l e and showed a d ro p in pH from 8*0 t o 6 .8 fo r
an averag e p e r io d of 20 h o u rs , w h e r e a s ,in th e l a t t e r ,
c o n ta m in a tio n had o c c u rre d a t the s t a r t , a n d t h i s medium
which was n o t changed had a pH o f a p p ro x im a te ly 7 .1 on t h e
n i n t h day.
On August 8 t h ,
34 worms
a c a t was s a c r i f i c e d , and 32 t o
were t a k e n from t h e i n t e s t i n e . These
£ . t a e n l a e f o r m i s d id n o t
ov er t h e
respond w ell t o th e Tyrode s o l u t i o n
t r y p t o n e - d i g e s t a g a r to which a t r a c e o f b i l e s a l t
had been added.
d a ily .
a d u lt
These worms were changed t o f r e s h media
Only one worm o f t h e l a r g e number u sed was a l i v e a t
th e end o f 77 h o u rs .
T a b le IV i s a c o m p ila tio n of th e r e s u l t s of 20
ex p e rim e n ts in m a in ta in in g l a r v a e and a d u l t T . t a e n l a e f o r m i s
a l i v e i n a e r o b ic c u l t u r e m edia.
I n g e n e r a l, th e sim p le
s a l t s o l u t i o n s su ch a s Tyrode seem more e f f e c t i v e th a n any
o f th e more complex c o m b in a tio n s .
Data f o r l o n g e v i t y o f
t h e worms were based upon o b s e r v a t io n o f m o t i l i t y i n e v e ry
c a se .
One a tte m p t a t a e ro b ic c u l t u r e u s in g c a t serum
was made i n November 1941.
F ive t e s t tu b e s were s e t u p . To
each o f f o u r t u b e s , 10 c c . o f m o dified Tyrode s o l u t i o n (no
d e x t r o s e was added and to two of t h e s e an a d d i t i o n a l one cc.
o f c a t serum was added.
added t o t h e f i f t h t u b e .
Ten cc. o f mammalian R in g er was
One or two l a r v a e were p la c e d i n
-5 0 -
ea ch o f th e t u b e s .
The r e s u l t s a r e re c o rd e d i n T ab le V.
TABLE V. A COMPARISON OF VITALITY OF LARVAE OF
TAENIA TAENIAEFORMIS IN MODIFIED TYRODE (NO DEXTROSE)
________ fflTH AND fflTHOPT UNHEATED CAT SERUM________
Medium
No. o f Worms
Tyrode w ith c a t serum
Tyrode w ith o u t serum
Tyrode w ith c a t serum
Tyrode w ith o u t serum
Mammalian R in g er s o l u t i o n
2
2
1
1
1
S u r v iv a l a s i n d i c a t e d
bv m o t i l i t y _____
24
73
8
48
96
h o u rs
h o u rs
h o u rs
h ours
h o u rs
3 . C u ltu r e E xperim ents w ith P e r f u s i o n A pparatus
A ' g r a v i t a t i o n a l p e r f u s io n a p p a ra tu s was c o n s t r u c t e d .
A r e s e r v o i r was p la c e d above th e i n c u b a t o r .
G la ss tu b in g l e d
i n t o th e in c u b a to r and co n n e cted w ith a minimum o f s i x f e e t
o f c o i l e d g l a s s tu b in g i n th e top o f th e i n c u b a t o r .
The
medium used was n e x t c a r r i e d i n t o t h e c u l t u r e f l a s k ( E r l e n meyer) and th e flo w was r e g u l a t e d so t h a t t h e f l u i d dropped
on th e s u r f a c e o f t h e sh a llo w s o l u t i o n c o n t a i n i n g th e l a r v a e
o f 1 . ta e n la e fo rm is.
By r e g u l a t i n g th e h e ig h t from which
t h e f r e s h medium d ro p p e d , and th e d e p th o f th e medium con­
t a i n i n g th e worms, a c e r t a i n amount o f d i s t u r b a n c e cou ld be
m a in ta in e d i n th e l a t t e r medium.
When s e v e r a l worms were
p r e s e n t i n a sm all f l a s k , t h e y had a tendency t o knot and
c o i l i n t o a compact mass.
T h is was l a r g e l y p re v en ted by
p l a c i n g a l a y e r o f s t e r i l e g l a s s beads o v er t h e bottom o f
th e c u ltu re fla s k .
The o v e rflo w tu b e was a sip h o n which could
-5 1 -
be r e g u l a t e d so t h a t t h e d e p th o f t h e medium i n t h e c u l t u r e
f l a s k was c o n t r o l l e d .
L a te r , a m o d ific a tio n o f t h i s " s in g le -
f la s k * p e r f u s i o n s e t - u p was c o n s tr u c te d so t h a t t h r e e c u l ­
t u r e f l a s k s were u sed i n a s e r i e s .
T h is was done becau se i t
was c o n s id e re d t h a t some a c c l i m a t i z a t i o n o f t h e medium by
t h e p r e v io u s p re se n c e o f worms m ight make i t more f a v o r a b le
f o r i s v i t r o m ain ten an ce .
T ab le VI g iv e s th e r e s u l t s o f
p e r f u s i o n ex p e rim e n ts w ith t h e p r e v io u s ly d is c u s s e d " o c c a s i o n a l change" method as a c o n t r o l .
TABLE VI. A COMPILATION OF THF RESULTS OF PERFUSION
EXPERIMENTS WITH TAPEWORM LARVAE COMPARED WITH THE METHOD
_____________ OF OCCASIONAL CHANGE OF MEDIUM
D ate
May 1941
Medium
Method
S u r v iv a l
i n Hours
one b o u i l l o n cube p e r
sin g le ­
l i t e r o f 1% d e x t r o s e fla sk
Mam. R ing er w ith a t r a c e P e r f u s i o n
29
o f sodium t a u r o c h o l a t e
o c c a sio n a lchange
72
May 1941 1!? d ex tro se-m am .R in g er
sin g le ­
fla sk
P erfu sio n
99
se rie s-fla sk
P e r f u s i o n (A) 71
(E) 36
(C) 56
Tyrode w ith o u t d e x tr o s e o c c a s i o n a l change
113
1% d ex tro se-m am .R in g er
sin g le -fla sk
P erfu sio n
107
June 1941 1% dex tro se-m am .R in g er
sin g le -fla sk
P e rfu sio n
78
S e rie s-fla sk
P e r f u s i o n (A) 58
,B) 51
>C) 30
-5 2 -
4 . A naerobic C u ltu r e E xperim ents
A number o f e x p e rim e n ts were t r i e d u t i l i z i n g modif i e d - a n a e r o b i c and a n a e ro b ic c u l t u r e methods.
f o u r t e s t tu b e s were s e t up.
In March 1941,
Each c o n ta in e d 10 c c . o f mam­
m alian R inger s o l u t i o n w ith g l u c o s e .
N othing was added to
t h e f i r s t tu b e , b u t t r a c e s of u r e a , a l l a n t o i n , and u r i c a c id
were added t o th e se c o n d , t h i r d , and f o u r t h tu b e s r e s p e c t i v e l y .
A f t e r one norm had been p la c e d i n each t u b e , N ujol was added
o v e r th e R inger so t h a t t h e t e s t tu b e was n e a r l y f i l l e d .
Then ru b b e r s t o p p e r s were i n s e r t e d t i g h t l y i n t h e s e fo u r
tu b e s.
I t was c o n s id e re d t h a t th e worm would be i n a medium
which was n e a r l y a n a e r o b ic a f t e r i t had u t i l i z e d oxygen a l ­
re a d y p r e s e n t .
A second s e t o f tu b e s u s in g s i m i l a r media
was s e t u p , b u t th e s e c o n t r o l s were cotton-pluggedjand n o t
covered w ith N u jo l.
F iv e days a f t e r th e b eg in n in g o f t h i s
e x p e rim e n t, a l l worms showed m o t i l i t y , b u t th e a e r o b ic s e r i e s
appeared to b e i n b e t t e r c o n d i t i o n and a s a group showed
g r e a t e r a c t i v i t y th a n th o s e o f th e a n a e ro b ic s e r i e s .
A ll
worms were changed t o f r e s h media a t t h i s tim e , but newly
a u to c la v e d R in g er was u sed w ith th e a n a e ro b ic s e r i e s i n an
a tte m p t t o keep oxygen c o n te n t to a minimum.
Only th e worms
i n g lu c o s e and g lu c o s e - u r e a s o l u t i o n s were a l i v e on th e
s ix th day.
In November 1941, f r e s h l y ground t i s s u e from a h a l f ­
grown k i t t e n which was n o t i n f e c t e d with a d u l t T aenia
-5 3 -
t a e n i a e f o r m l s . were p la c e d i n th e low er h a l f o f t e s t tu b e s
a s f o llo w s :
I.
II.
III.
IV .
V.
VI.
V II.
V III.
I n t e s t i n e - m u s c l e i n f u s i o n (T ry p s in
L iv e r-m u s c le i n f u s i o n
"
Muscle i n f u s i o n
"
Stom ach-Spleen-M uscle i n f u s i o n »
and NaHCO* pH
*
it «-■
"
"
w
w
Stom ach-Spleen-M uscle I n f u s i o n (P e p s in and HC1
Muscle i n f u s i o n
"
"
"
L iv e r-m u sc le i n f u s i o n
"
"
«
in te stin e -m u sc le in fu sio n
n
u
n
7-9)
«
n
n
pH 1 -4 )
"
«
n
The m a t e r i a l I n each tu b e was covered w ith mammalian
R in ger s o l u t i o n .
One to t h r e e worms removed from t h e i r c y s t s
were p la c e d i n each t e s t t u b e , e x c e p t No. VI which had t h r e e
c y sts.
One c y s t was a l s o p la c e d i n t e s t tu b e s I and I I . A
t r a c e of b i l e s a l t was added t o each tube o f t h e a l k a l i n e
se rie s.
Movement o f worms could n o t be d is c e r n e d i n any o f
t h e t e s t tu b e s a f t e r th e f i r s t few h o u r s .
None o f t h e worms
were l i v i n g on t h e s i x t h d ay , when t h e l a r v a e were removed
from t h e a n a e ro b ic tu b e s and c a r e f u l l y examined.
However,
th e worms o f t h e a c i d s e r i e s , b o th when removed from c y s t s
and w ith in th e c y s ts ,w e r e i n t e t t e r c o n d i t i o n t h a n were th o s e
o f the a l k a l i n e s e r i e s .
The a n a e ro b ic medium o f Brewer (1940) was u t i l i z e d
i n some e x p e rim e n ts.
T his c o n ta in e d pork i n f u s i o n s o l i d s ,
p e p to n e , and d e x t r o s e as food s o u rc e s and was b u f f e r e d a t a
pH o f 7 . 5 .
S ix in c h t e s t t u b e s , which were t h r e e - f o u r t h s o f
an In c h i n d ia m e te r were u t i l i z e d and th e s e were f i l l e d to
a h e ig h t o f seven c e n tim e te r s which was about 15 c c . o f b r o t h .
-5 4 -
F iv e t e s t tu b e s were s e t up and two to f o u r sm a ll l a r v a e
were p la c e d i n each tu b e .
M o t i l i t y i n th e worms was ob­
se rv e d a f t e r 27 h o u rs i n t h i s medium, a lth o u g h i t was r e ­
duced i n th e a n a e ro b ic m edia.
S in ce much a g i t a t i o n o f th e
t e s t tu b e i n c r e a s e d t e m p o r a r ily
t h e amount o f oxygen i n th e
b r o t h , a tte m p ts to in d u ce movement o f t h e worms i n a n a e ro b ic
s e r i e s were r e s t r i c t e d to g e n t l e sh ak in g o f t h e c o n t a i n e r s .
November 22, a n o th e r s e r i e s was s e t up u t i l i z i n g
t h i s a n a e ro b ic medium.
Two t o f o u r worms w ere p la c e d i n each
o f f o u r t e s t t u b e s , w hile In th e f i f t h two c y s t s c o n ta in in g
worms were u s e d .
M o t i l i t y was observed a f t e r ab o u t f i v e h o u rs ,
b u t su b seq u en t o b s e r v a t i o n f a i l e d t o n o te worm a c t i v i t y .
Two o f t h e tu b e s i n t h i s s e r i e s dev elo p ed a l o c a l i z e d p r e c i p i ­
t a t e suspended around th e p a r a s i t e s .
a n a e r o b ic b a c t e r i a l grow th.
T h is p ro b a b ly I n d i c a t e s
A lthough t h e worms were dead
on th e f i f t h day when th e y were removed from th e t e s t tu b e s
and exam ined, th e y a l l ap peared to be i n good c o n d i t i o n , ex­
c e p t f o r a g ra y n e s s of t h e body.
I n March 1942, two sm all worms were p laced i n a
t e s t tu b e which c o n ta in e d a s h o r t e r column o f a n a e ro b ic
b r o t h , s i n c e , i f th e s e p a r a s i t e s p o s s e s s e d a fa c u lta tiv e metab­
o lis m , an in c o m p le te ly a n a e ro b ic environm ent m ight be more
b e n e fic ia l.
About two c u b ic c e n tim e te r s o f c h ic k embryonic
e x t r a c t were added to t h i s medium.
A few days l a t e r , th e s e
two worms were p la c e d I n a c o tto n -p lu g g e d E rlenm eyer f l a s k
-5 5 -
i n mammalian R inger s o l u t i o n .
The m ethylene b lu e which
th e y had d e r iv e d from th e a n a e ro b ic medium tu rn e d th e worms
b lu e when th e y were removed from th e c u l t u r e tu b e s and ex­
posed t o oxygen o f th e a i r .
T h is I n d ic a te d t h a t th ey were
i n an a c t u a l a n a e ro b ic environm ent in the t h i o g l y c o l l a t e
a n a e ro b ic medium, even w ith th e d i l u t i o n invoked by t h e ad­
d i t i o n of c h ic k embryonic f l u i d .
P a rt I I . R e sp ira tio n stu d ie s
I n F eb ruary and March 1940, a s e r i e s o f d eterm in ­
a t i o n s were made on t h e r a t e o f oxygen consum ption by l a r v a e
o f T aenia t a e n l a e f o r m i s .
A f t e r s e v e r a l s e r i e s o f p r e li m i n a r y
e x p e rim e n ts to d e v e lo p te c h n iq u e , a r a t was k i l l e d and
e le v e n worms removed from t h e l i v e r .
Seven o f t h i s group
were p la c e d in mammalian R inger s o l u t i o n .
A f te r about two
h o u r s , th ey were t r a n s f e r r e d to t h e r e s p i r a t o r y chamber
(30 m l. hypodermic s y r i n g e ) .
A two c u b ic c e n ti m e t e r sample
was t a k e n im m ed iately i n t o th e r e a c t i o n chamber (10 m l.
hypodermic s y rin g e ) and t e s t e d f o r oxygen.
T his gave th e
i n i t i a l oxygen v a lu e f o r t h e s o l u t i o n .
At t e n m inute i n t e r ­
v a l s , two c c . sam ples were withdrawn.
These were t e s t e d f o r
oxygen and th e r e s u l t s re c o rd e d .
S u b se q u e n tly , t h e worms
were p la c e d in t h e r e s p i r a t o r y chamber in 0 .0 0 0 2 N KCN made
up i n mammalian R in g e r s o l u t i o n .
The r e s u l t s o f t h e s e two
s e r i e s o f r e s p i r a t i o n s t u d i e s a r e ta b u l a t e d In T a b le s VII
-5 6 -
and V I I I .
These t a b l e s show th e a n a ly s e s o f r e s u l t i n g
cu rv e s when th e amount o f oxygen u sed i s p l o t t e d a g a in s t
t h e tim e i n t e r v a l .
F ig u re 1.
These d a t a a r e compared i n th e graph i n
T h is c o n c e n t r a t i o n o f KCN showed no e f f e c t upon
t h e r e s p i r a t i o n o f t h e s e worm3 as I s e v id e n t from i n s p e c t i o n
o f th e g ra p h .
On c o n c lu s io n o f th e ex p e rim e n t, t h e worms
were p la c e d i n mammalian R ing er s o l u t i o n a t 37° C.
A few
h o u rs l a t e r , t h e i r consum ption o f oxygen was measured by
a n o th e r run i n norm al mammalian R in g er s o l u t i o n .
About two
hours l a t e r , th e y were p la c e d In 0.0001 N KCN-Ringer s o l u t i o n ,
and i t s e f f e c t on oxygen consumption was measured ( F ig . 2 ) ,
a f t e r which th e worms were p la c e d i n mammalian R in g er s o l u ­
t i o n and r e t u r n e d to th e i n c u b a t o r u n t i l tim e f o r th e n e x t
s e r i e s , a p ro c e d u re t h a t was fo llo w ed th ro u g h o u t.D a ta f o r
F ig u re 2 a r e a n a ly se d i n T a b le s IX and X.
F ig u re 7 i s a
com posite o f F ig u re s 1 and 2 and r e v e a l s t h a t w h ile t h e r e
p ro b a b ly i s no Im m ediate e f f e c t o f cyanide on r e s p i r a t i o n
a t any c o n c e n t r a t i o n s u s e d , y e t t h e r e may have been a d elay ed
e f f e c t from th e 0 .0 0 0 2 N KCN-Ringer s o l u t i o n as i n d i c a t e d by
th e s l i g h t l y low er s lo p e o f c u rv es 3 and 4 i n t h i s F ig u r e .
F ig u r e 3 compares th e c u rv e s o f oxygen consum ption
o f th e s e worms i n mammalian R in g er and i n 0 .0 0 5 N KCN-Ringer
s o l u t i o n s , and t h e s e d a ta a r e compiled and a n a ly se d i n T ab les
XI and X II .
The measurem ents o f oxygen consum ption 7/1 t h
mammalian R in g er s o l u t i o n were made n in e h o u rs a f t e r th e
-5 7 -
0.0001 N KCN s e r i e s o f F ig u re 2.
Measurements w ith
0.005 N KCN-Ringers follow ed Im m ed iately .
F ig u re 3 shows
t h a t t h e e f f e c t o f t h i s c o n c e n t r a t i o n o f cy an id e upon th e
r a t e o f oxygen consum ption i s pronounced.
T h is o b s e r v a t io n
p r e s e n t s ev id en c e t h a t a t l e a s t a p o r t i o n o f th e r e s p i r a t i o n
o f theseworms i s c y a n i d e - s e n s i t i v e . The r e s u l t s p l o t t e d i n
F ig u r e 4 and a n a ly se d I n T a b le s X III and XIV compare th e e f ­
f e c t o f mammalian R in g er s o l u t i o n w ith 0 .0 0 2 N KCN-Ringers
upon t h e r e s p i r a t i o n of th e s e l a r v a e 60 hours a f t e r rem oval
from t h e h o s t and a p p ro x im a te ly 24 hou rs a f t e r t h e m easure­
ment w ith 0 .00 5 N KCN-Ringer s o l u t i o n .
F ig u r e s 5 and 6 com­
p are t h e e f f e c t on t h e s e l a r v a e o f 0.0015 N and 0 .0 01 N
KCN-Ringer s o l u t i o n s w ith mammalian R in g e r, 90 and 157 h ou rs
r e s p e c t i v e l y a f t e r removal from th e r a t h o s t .
f i g u r e s are a n a ly se d i n T ab les XV to X V III.
D ata f o r th e s e
Each o f t h e s e
t h r e e c o n c e n t r a t i o n s o f cy an id e caused a pronounced drop
i n t h e r a t e o f r e s p i r a t i o n as compared w ith th e r a t e in
mammalian R inger s o l u t i o n .
I t should be n o ted t h a t oxygen consum ption was
m easured f i r s t i n mammalian R in g er s o l u t i o n fo llo w ed immedi­
a t e l y by m easurements I n th e c y e n id e -R in g e rs .
Worms were
k e p t i n s t e r i l e , mammalian R in g er s o l u t i o n a t 3 7 .5 ° c e n tig r a d e
u n t i l th e n e x t tim e u s e d .
No d e x tr o s e wss p r e s e n t i n t h i s
R in g er s o l u t i o n a t any tim e .
F ig u re 8 i s a com posite o f f i g u r e s 3 and 4.
It
-5 8 -
seems to show t h a t th e e f f e c t o f cy a n id e i s r a t h e r prolonged
i n t h e c a s e o f th e s e l a r v a e .
The c o n c e n t r a t i o n o f cyanide
used f o r t h e d a t a o f F ig u re 3 was 0 .0 0 5 norm al,end t h i s
c o n c e n t r a t i o n seems to be a f f e c t i n g th e r a t e o f r e s p i r a t i o n
i n mammalian R in g er s o l u t i o n 24 h o u rs l a t e r a s shown by i t s
l e s s e r s lo p e when compared w ith r e s p i r a t i o n on mammalian
R in g e rs a t th e 36 hour p e r io d .
T h is same d elay ed e f f e c t of
t h e cy an id e c o n c e n t r a t i o n u sed i n t h e Im m ediately p re c e d in g
s e r i e s i s c o n s ta n t th ro u g h o u t th e e n t i r e ex perim ent so t h a t
F ig u re 8 i s r e p r e s e n t a t i v e .
I t i s e v id e n t t h a t some r e ­
covery from th e e f f e c t o f cyanide i s o c c u r r i n g , b u t u n d e r
t h e c o n d itio n s o f t h e e x p e rim e n t,tim e s u f f i c i e n t f o r com plete
re c o v e ry had n o t been a llo w e d .
F ig u re 9 shows c o m p o site ly th e d a t a which were
shown g r a p h i c a l l y i n F ig u r e s 5 and 6 .
The r a t e o f oxygen
consum ption i n mammalian R inger s o l u t i o n a t 157 hours i s
low er th a n th e c o rre s p o n d in g r a t e a t 90 h o u rs , b u t t h i s
e f f e c t a g a in i s due c h i e f l y to th e cy an id e c o n c e n t r a t i o n
employed i n th e jle a rlie r s e r i e s , i n t h i s c a se 0.0015 normal
c y a n id e .
That th e e f f e c t i s n o t due e n t i r e l y t o "age" o f
th e l a r v a e i s r e v e a le d by n o tin g t h a t t h e r a t e o f r e s p i r a t i o n
o f t h e same worms i n mammalian R inger s o l u t i o n a t 288 hours
(T a b le XIX) I s h ig h e r th a n t h e r a t e a t e i t h e r 90 o r 157 h o u rs ,
and t h i s f a c t shows t h a t th e e f f e c t o f cy a n id e had worn o f f
to a l a r g e e x t e n t .
The f i n a l m easurements o f oxygen consump-
-5 9 -
t i o n were ta k e n a t th e i n v i t r o age o f 364 h o u rs (T a b le XX)
f o r t h i s group o f worms, and th e low er r a t e o f oxygen con­
sum ption h e re I s due to t h e f s c t t h a t t h e worms a r e ”o l d n
from t h e s t a n d p o i n t o f j j i v i t r o c u l t u r e .
The r e s u l t s p l o t t e d i n F i g u r e s 10 and 11 and
a n a ly s e d i n T a b le s XXI - XXIV, s e rv e a s a check f o r th e p r e ­
v io u s com p ariso n s,
i n t h i s c a s e , 24 worms were u t i l i z e d ,
b u t t h e i r t o t a l weight was s l i g h t l y more th a n o n e - t h i r d t h a t
o f th e seven worms i n t h e p re v io u s s e r i e s , whose r e s p i r a t i o n
was p l o t t e d i n t h e e a r l i e r c h a r t s .
Comparison w ith F ig u r e s 5
and 6 i n which th e cyan id e c o n c e n t r a t i o n s a r e com parable,
shows t h a t t h e e f f e c t o f cy an id e upon t h e r a t e o f r e s p i r a t i o n
i s v e ry s i m i l a r f o r t h e two groups o f worms.
In d e e d , th e
c u rv e s show t h a t th e oxygen consum ption was d e p re sse d much more
by th e 0 .0 0 1 5 N t h a n by th e 0.001 N c o n c e n t r a t i o n o f c y a n id e ,
and i t a p p e a rs t h a t t h e form er c o n c e n t r a t i o n I s n e a r t h e
c r i t i c a l end p o i n t , where t h e r e s p i r a t i o n o f t h e s e worms i s
f i r s t m arkedly a f f e c t e d by t h i s p o is o n .
A s e r i e s o f e x p e rim e n ts on f r e s h , whole l a r v a e o f
T aenia t a e n i a e f o r m i s was conducted to d e te rm in e w hether m ethylene
b lu e i s reduced to le u c o -m e th y le n e b lu e by t h e s e worms i n
th e a b s e n c e o f oxygen.
S t e r i l e p r e c a u t i o n s were o b s e rv e d .
From one t o f i v e l a r v a e were t e s t e d i n v a r io u s e x p e rim e n ts
i n which s ta n d a r d Thunberg tu b e s were u s e d .
u a te d by means o f an a s p i r a t o r .
These were evac­
M easurements o f t h e c o lo r
-6 0 -
change i n m eth y len e b lu e were ta k e n e l e c t r o m e t r i c a l l y .
R e d u c tio n o f m ethylene blue o c c u rre d when th e worms were
s t u d i e d i n normal mammalian R in g er s o l u t i o n ( F i g . 1 2 ).
R e d u c tio n of m ethylene b lu e o c c u rre d a l s o when th e R inger
S o l u ti o n c o n ta in e d one p e r c e n t d e x tr o s e (F ig . 1 3 ).
But
i n s p e c t i o n o f F ig u r e 14 w i l l r e v e a l t h a t th e p re s e n c e o f
g lu c o s e as a s u b s t r a t e d id n o t i n c r e a s e th e speed o f
m eth y len e b lu e r e d u c t i o n .
T a b le s XXV - XXVIII.
These c u rv e s a r e a n a ly s e d i n
The l a r v a l tapeworms u sed I n th e s e
e x p e rim e n ts were n e a r l y seven months of ag e, so t h a t th e y
p ro b a b ly were "m atu re” , and i n f e c t i v e f o r th e f i n a l h o s t .
One group o f la r v a e ,a p p r o x i m a t e l y t h r e e months o f age,w ere
removed from a r a t h o s t and s tu d ie d by means o f t h e
"Thunberg T echnique" u s in g mammalian R inger s o l u t i o n w ith ­
o u t d e x tro se .
M ethylene b lu e r e d u c t i o n by t h e s e worms I s
p l o t t e d i n F ig u re 15, and th e cu rv es a l l r e v e a l an e a r l y
la g .
I t seems p ro b a b le t h a t t h e r e i s a dehydrogenase
p r e s e n t which i s c a u s in g m ethylene b lu e r e d u c tio n and t h a t
i t i s a c t i n g upon some s u b s t r a t e o t h e r th e n g lu c o s e .
A f te r co m p letio n o f ex p e rim e n ts w ith ev acu ated
Thunberg t u b e s , l a r v a e were a c t i v e and i n good c o n d it i o n
when r e t u r n e d to a e r o b i c media.
TABLE I . A COMPAJRISON OP CHANGES IN THE pH
OF TYRODE*S SOLUTION CONTAINING TAENIA TAEHIAEFORMIS LARVAE
Medium
Amount
S t a r t i n g D ate, December 2,1938
T y ro d e ^ s o lu tio n
20 c c .
4 P.M.
I n i t i a l pH (Notes low ered w ith
HC1 i n t h i s s e r i e s . )
C o n ta in e r l
1 worm
Date and Hour E lap sed I n i t i a l pH o f Change
pH
o ld
in
o f change
tim e i n
cH
t o new media
h o u rs
_ media
Dec 3
5
5
6
6
7
8
9
9
10
10
11
11
12
12
13
18
14
15
16
16
17
17
18
19
C o n ta in e r I I
3 worms
pH o f Change
in
o ld
dH
media
24
below
4pm
8 .8
2 .0 ?
—
—
9 am
41
8 .8
2 .0 ?
6 .8
—
6pm
9
8 .8
—
—
—
—
9 am
15
8 .8
—
9
6pm
8 .8
1 .4
7 .4
12M
18
8 .8
0 .5
8 .3
6 .7
2 .1
22 30pm
2 6 .5
9 .0
8 .0
6 .5
2 .5
1 .0
24
2:30pm
9 .0
8 .4
0 .6
6 .9
2 .1
—
11pm
8 .9
8 .5
8 .0
0 .9
10:30am 1 1 .5
8 .3
0 .3
6 .6
7 .4
1 .2
5pm
6 .5
0 .9
8 .8
8 .8
0 .0
7 .9
10am
17
8 .9
8 .7
0 .2
1 .7
7 .1
12
10pm
8 .8
8 .7
0 .1
7 .4
1 .4
17
3pm
8 .9
8 .9
0 .0
7 .0
1 .9
10s30pm
7 .5
8 .8
8 .7
0 .1
7 .7
1 .1
2 s 30pm
16
9 .0
9 .0
0 .0
7 .7
1 .5
7 .5
9 .3
10pm
9 .3
0 .0
9 .2
0 ,1
11i30am 1 3 .5
8 .5
8 .8
0 .3
7 .9
0 .9
3pm
27.5
0 .2
8 .6
8 .4
6 .8
1 .8
deed
10am
19
8 .7
deed
6 .8
1 .9
6 : 30pm
8 .5
8 .6
7 .1
1 .5
11am
16
8 .4
* 7 .2
1 .2
7pm
8
8 .4
7 .6
0 .8
no r e a d i n g s , h at one worm i n C o n ta in e r I I s t i l l a l i v e
4pm
45
8 .6
dead
6 .7
Note:*Two o f th e t h r e e worms i n C o n ta in e r I I were dead a t
t h i s tim e .
TABLE I I . A STUDY OF TWO GROUPS OF JL. TAFNTAF.FORMTS
LARVAE, SHOWING THE pH CHANGES OCCURRING WITH TIME IN A
MODIFIED TYRODE SOLUTION.________________
Medium
Amount Used 20 c c .
M odified T yrode*s u t i l i z i n g
DIsodium p h o sp h a te I n s t e a d
o f Sodium b i c a r b o n a t e .
S t a r t i n g D ate:
December 15,1938
3 pm.
i n i t i a l pH 7 .6 e x c e p t i n t h e c a s e s n o ted where I t was low ered
by th e a d d i t i o n o f HC1.___________________________________________
C o n ta in e r I
C o n ta in e r I I .
Date and Hour E la p se d I n i t i a l 3 worms ( l sm all)
5 worms
o f change
tim e i n
pH
pH o f
change
pH o f change
t o new media
h o u rs
o ld
in
o ld
in
____________________________________ media_____ pH_____ media
pH
—
7 .6
D ec.15
3pm
11pm
8
7
.6
15
11
* 8 .4
10 am
16
6 pm
7 .6
8
16
17
7 .6
11am
17
7 .4
17
7 pm
8
4pm
21
7 .6
18
3pm
23
7 .6
19
7
* 8 .4
19
10pm
5pm
7 .6
20
19
5pm
21
24
7 .6
5pm
7 .6
22
24
4pm
23
7 .6
23
—
11am
43
25
4pm
27
53
One worm i n C o n ta in e r I I was
a f t e r the s t a r t o f the s e r i e s .
6 .2
7 .2
6 .4
6 .1
6 .4
6 .5
6 .0
7 .5
6 .2
6 .0
6 .0
6 .0
—1
I
a l i v e on December 27 -
6 .6
7 .3
6 .6
6 .5
6 .8
7 .2
6 .1
8 .0
* * 6 .4
6 .4
6 .5
6 .2
1 .0
1 .1
1 .0
1 .1
0 .6
0 .4
1 .5
0 .4
1 .2
1 .2
1 .1
1 .4
1 .4
1 .2
1 .2
1 .5
1 .0
1 .1
1 .6
0 .9
1 .4
1 .6
1.6
1 .6
I
293 ho urs
Note: *T his i n i t i a l pH o f 8 .4 was t h e re a d in g when a b ic a rb -T y ro d e * s
was s u b s t i t u t e d .
**The sm a ll worm was dead on Dec. 2 0 th . The rem ainin g worms
were a l i v e a t th e tim e o f t h e Dec. 23rd r e a d in g , a
minimum o f 197 h o u rs .
TAELE I I I . A STUDY OF THE pH CHANGES IN A VIEDIUH
CONTAINING UREA. FOUR LARVAE OF T- TARN1 AEFOPMIS # ERE
USED AS A GROUP AT FIRST, BUT LATER THE? WERE SEPARATED
______INTO GROUPS OF TWO
_________________
Date and Hour E lapsed I n i t i a l
pH o f
Change
o f change tim e In
pH
old
in
to nett .med i a h o u rs_______ _________1
--------------------ES__
Mar 13
13
14
14
15
15
15
16
4pm
10pm
10am
10pm
10am
4pm
10pm
10 am
4pm
16
10pm
16
10am
17
4pm
17
11pm
17
3pm
18
10pm
18
11am
19
5pm
19
3pm
20
9:30pm
20
4pm
21
6
12
12
12
6
6
12
8.4
8.4
8 .4
8.4
8 .4
8 .4
8 .4
8.4
6
6
12
6
7
16
7
13
6
22
6 .5
1 8 .5
8.4
8.4
8.4
8.4
8.4
8.4
8.4
8.4
8.4
8 .4
8 .4
8.4
—
—
—
2.2
6.2
2.8
5.6
3.4
5.0
5.1
3.5
2.4
6.0
2.9
5.5
.5 .1
3.5
Group I iGroup I I Gr bup I Group
2 .0
6 .4
2 .2
6.2
2
.
0
2 .2
6 .4
6.1
3
.
4
5 .0
3.5
4.9
3
.
3
5.5
3.1
5 .1
3 .5
4 .9
4 .9
4.4
3 .8
4 .6
3 .9
4.5
3 .2
2 .4
5 .2
6.0
3 .7
4 .7
2.4
6.0
2 .4
6 .0
1.7
6.7
3 .8
4 .6
3.6
4.6
1
.6
6 .8
2.3
6.1
3
.
7
4 .7
3 .0
4.6
TABLE IV.
A COMPILATION OF THE RESULTS OF EXPERIMENTS IN
WHICH LARVAE AND ADULTS OF TAENIA TAENIAEFORMIS
______________________ HAVE BEEN MAINTAINED ALIVE.___________
Medium
Date
S u r v iv a l I n Hours
LARVAE
J a n . 1938
A p r i l 1938
A p r i l 1938
A p r i l 1938
O ct. 1938
Nov. 1938
Dec. 1938
Dec. 1938
March 1939
March 1939
A p r il 1939
A p r il 1939
May 1939
May 1939
Ju n e 1939
5 p e r c e n t T ry p to n e i n
d i s t i l l e d w ater
5 p e r c e n t T ry p to n e ,a n d
Mammalian R in g e r w ith
m acerated i n f e c t e d l i v e r
Mam. R inger s o l u t i o n p lu s
in fe c te d l i v e r d ig e st
Tyrode s o l u t i o n
"
”
p lu s 0.0 1
p e r c e n t p henol
Tyrode s o l u t i o n
it
n
March 1940
48
324
324
156)k i l l e d by
5 0 )o v e r h e a tin g
211
Tyrode o v er p e p t o n e - d ig e s t
a g a r 2% w ith a c r i f l a v i n e
Mam. Ringer S o l u t i o n
Mam. R ing er p lu s h e x y lr e s o c r i n a l ( S . T. 37)
M ostly Mam.Ringer s o l u t i o n
tt
tt
May 1940
120
m o d ifie d Tyrode—double
sodium b ic a r b
384
mod. Tyrode —disodium
p h o sp h ate r e p la c e d b i c a r b 288
Tyrode - d ouble b i c a r b p lu s
168
0 .0 1 p e r c e n t u r e a
n
n
tt
ft
it
ti
192
Tyrode over P e p to n e d l g e s t ag a r
120
Disodium phos. Tyrode o v e r
in fe c te d -ra t-liv e r-d ig e st
i n g e l a t i n e and a g a r
120
Tyrode over p l a i n a g a r 2% 144
n
" p e p to n e - d ig e s t
144
agar
Tyrode o v er t r y p t o n e - d i g e s t 100
a g a r 1% w ith b i l e s a l t s
169
August 1939
August 1939
144
n
it
it
ii
if
it
Mam.Ringer w ith t r a c e o f
G lucose
48
48
161
161
1 6 8 )i t was n o t
288)n o ted when
1 2 0 ;t h e s e d ie d .
576
ADULT
Aug. 1939
Tyrode ov er t r y p t o n e - d i g e s t
a g a r 1% w ith b i l e s a l t s
77
TABLE V II. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF SEVEN T. TAENIAEFOBMIS LARVAE IN MAMMALIAN
RINGERS,TAKEN ABOUT TWO HOURS AFTER REMOVAL FROM THE RAT.
THESE LARVAE WEIGHED 1 .5 GM. APPROXIMATELY.________________
E lap sed
tim e i n
m inutes
In itia l.a
10
80
30
40
50
Am* t .
02
Thio E quiv.
used
c c /L .
cc.
2 .0
5 .6
1 .6
1 .4
1 .3
1 .0
0 .8 5
4 .4 8
3 .9 2
3 .6 4
2 .8 0
2 .3 8
A c tu a l
02
u p ta k e
( a-x )
1 .1 2
1 .6 8
1 .96
2 .80
3 .2 2
C a lc .
02
u p ta k e
1 .0 2
1 .6 2
2 .2 1
2 .8 1
3 .4 0
Formula
y • mi t b
m
b
y
x
«
«
.0595
.43
(a -x )
m inu tes
TABLE V I I I . SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME SEVEN LARVAE IN 0.0002 N KCN-RINGERS,
TAKEN ABOUT FIVE HOURS AFTER REMOVAL FROM THE RAT.____________
E la p se d
tim e i n
m in utes
Am* t .
°2
T h io . E qulv.
c c /L .
used
cc.
2 .1 5
10
20
30
40
50
1 .8
1 .5
1 .3
1 .1
0 .8
A ctu a l
°2
u p ta k e
(a -x )
C a lc .
Oo
u p ta k e
Formula
y «
m
f b
ju g *
5 .0 4
4 .2 0
3 .6 4
3 .0 8
2 .2 4
0 .9 8
1 .8 2
2 .3 8
2 .9 4
3 .7 8
1 .0 8
1 .7 1
2 .3 4
2 .9 7
3 .6 0
m x .063
b • .45
TABLE IX. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME SEVEN LARVAE IN MAMMALIAN RINGERS,
TAKEN ABOUT SO HOURS AFTER REMOVAL FROM THE BAT.____________
E la p se d
tim e i n
m in u te s
In itia l., a
10
£0
30
40
50
60
70
80
90
Am’ t .
Thio E qulv.
u se d
cc/L
cc.
1.7 5
5 .4 4
1 .9
1 .5
1 .3
1 .2 5
0 .8 5
5 .3 2
4 .2 0
3 .6 4
3 .5 0
2 .3 8
—
0 .7 5
0 .5
0 .4
—
2 .1 0
1 .4 0
1 .1 2
A c tu a l
02
u p ta k e
(a -x )
0 .1 2
1 .2 4
1 .8 0
1.94
3.06
-
3.3 4
4 .0 4
4 .3 2
C a lc .
02
u p ta k e
0 .5 4
1 .0 3
1 .5 3
2 .0 2
2 .5 2
3 .0 2
3 .5 1
4 .0 1
4 .5 0
Formula
y = mx-**b
m- 0.0496
b - ( - .0 4 )
TABLE X. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN 0.0001 N KCN-RINGERS.
READINGS WERE MADE ABOUT 22 HOURS AFTER REMOVAL FROM THE
RAT AND ABOUT TWO HOURS AFTER THE SERIES TABULATED IN
TABLE IX.
E la p se d
tim e i n
m inuted
Am* t .
°2
Thio E q uiv.
u sed
c c /L .
cc.
In itia ls, a
10
20
30
40
50
60
70
80
90
A ctu a l
°2
u p ta k e
(a -x )
C a lc .
°2
u p ta k e
Formula
y s mx + b
5 .6
1 .8 5
1 .6 5
1 .4 3
1 .2
1 .1
0 .9 5
0 .8
—
0 .5
5 .1 8
4 .6 2
4 .0 0
3.36
3 .0 8
2.66
2 .2 4
—
1 .4 0
0 .4 2
0 .9 8
1 .60
2 .2 4
2 .52
2 .9 4
3 .36
—
4.2 0
0 .5 3
0 .9 9
1 .4 5
1 .9 1
2.36
2 .8 2
3 .2 8
3 .7 4
4 .2 0
mr 0.0458
b s 0.0 7 5
TABLE X I.
SHOWING THE BESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN MAMMALIAN RINGERS ABOUT
26 HOURS AFTER REMOVAL FROM THE RAT._________________________
E la p se d
tim e i n
m in u te s
Am* t .
°2
Thio E q u lv .
u sed
c c /L .
cc,
I.ni;y.al= a
10
SO
SO
40
50
2 ,1
5 ,8 ?
1 .7
4.76
—
1 .8
0 .8
0 .6
—
S . 36
2.8 4
1 .68
A c tu a l
02
u p ta k e
(a -x ) _
1 .1 2
—
2 .5 2
3 .6 4
4 .2 0
C a lc .
02
u p ta k e
0 .9 8
1 .8 2
2.66
3.50
4 .3 4
Form ula
y-b. mx
1
m » 0 ,0 8 4
b : 0 .1 4
TABLE X II. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN 0 .00 5 N KCN-RINGERS,
IMMEDIATELY FOLLOWING THE SERIES TABULATED IN TABLE XI.
E la p se d
tim e i n
m in u te s
In itia l» a
10
20
30
40
50
60
70
Am*t.
02
Thio Equiv.
c c /L .
used
cc.
..
?«0
5.6
1 .9
1 .7
1 .6
1 .5
1 .2
1.15
1.0 0
5 .3 2
4.76
4 .48
4 .20
3.36
3.22
2.80
A c tu a l
02
u p ta k e
(a -x )
0 .2 8
0 .8 4
1 .1 2
1 .4 0
2 .2 4
2 .3 8
2 .8 0
C a lc .
02
u p ta k e
0 .4 2
0 .84
1.26
1 .6 8
2.10
2 .52
2 .9 4
Formula
y • mx
b
m-- 0 .0 4 2
b = 0 .0
TABLE X I I I . SHOWING THE RESULTS OF MEASUREMENT OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN MAMMALIAN RINGERS ABOUT
60 HOURS AFTER REMOVAL FROM THE RAT__________________________
E lap sed
tim e i n
m in u tes
In itia l» a
10
20
30
40
50
Am*t.
°2
Thio E qulv.
used
c c /L .
cc* _
1*7
1 .4
1 .2
0 .9
—
0.6
.
A c tu a l
02
u p ta k e
(a -x )
C a lc .
02
u p ta k e
Formula
y * mx t b
4.7£__
3 .9 2
3 .3 6
2 .5 2
—
1 .6 8
0 .8 4
1 .4 0
2 .2 4
—
3 .0 8
0 .8 1
1 .4 3
2 .0 4
2 .67
3 .2 7
m - .062
b » .195
TABLE XIV. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN 0 .0 0 2 N KCN-RINGERS
IMMEDIATELY FOLLOWING THE SERIES TAEULATED IN TABLE X I I I .
E la p se d
tim e In
m in utes
In itia l-a
10
20
30
40
50
60
70
Am* t .
02
Thio E qulv.
u sed
c c /L .
cc.
A c tu a l
°2
u p ta k e
_ (a_-x)
C a lc .
02
u p ta k e
Formula
y » mx-t- b
I t S ... ..S,Q4
1 .7
1.6
1 .5
1 .5
1 .3 5
1 .0
0 .9
4 .7 6
4 .4 8 .
4 .2 0
4 .2 0
3 .7 8
2 .8 0
2 .5 2
0 .2 8
0.56
0 .8 4
0 .8 4
1.26
2 .2 4
2 .5 2
0 .2 0
0 .56
0 .9 2
1 .2 8
1 .6 4
2 .0 1
2 .37
m- 0.0361
b * (-0 .1 6 )
TABLE XV. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN MAMMALIAN RINGERS ABOUT
90 HOURS AFTER REMOVAL FROM THE BAT._________________________
E lap sed
tim e I n
m inu tes
In itia l* a
10
20
30
40
50
60
Amf t .
°2
Thio E q u lv .
used
c c /L .
cc.
1 .6
4 .7 1
1 .6
1 .4
1 .0
1 .0
4 .4 8
3 .92
2 .60
o.e
0 .6 3
—
2 .2 4
1.76
A c tu a l
02
u p ta k e
(m^x)
0 .2 3
0 .7 9
1 .9 1
—
2 .4 7
2 .9 5
C alc.
°2
u p ta k e
0 .2 3
0 .7 8
1 .33
1 .8 8
2 .4 3
2.9 8
Formula
y a mx -t- b
m« 0 .055
b=. ( - 0 .3 2 )
TABLE XVI. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN 0.0015 N KCN-RINGER
IMMEDIATELY FOLLOWING THE SERIES TABULATED IN TAELE XV.
E lap sed
tim e i n
m inutes
Am* t .
Op
Thio Equlv.
used
c c /L .
cc.
I r i t i s - ? . 1 .9 5
10
20
30
40
50
60
70
80
1.75
1 .6 5
1 .6
1 .4
1 .3
1 .2
1 .1
0 .85
A c tu a l
°2
u p ta k e
farx )
C a lc .
Op
u p ta k e
Formula
y tr mx-/- b
5.46
4 .9 0
4 .6 2
4 .4 8
3 .9 2
3 .6 4
3.36
3 .0 8
2 .3 8
0 .56
0 .8 4
0 .9 8
1 .5 4
1 .8 2
2 .1 0
2 .3 8
3 .0 8
0 .47
0 .8 1
1 .15
1 .49
1 .8 3
2 .1 8
2 .5 2
2.86
m* 0 .084
b s 0 .1 3
TABLE XVII. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN MAMMALIAN RINGERS ABOUT
157 HOURS AFTER REMOVAL FROM THE RAT._________________________
E la p se d
tim e i n
m in u te s
in itia ls
10
20
30
40
50
60
70
80
Am* t .
°fi
Thio E qulv.
u sed
c c /L .
cc.
1^8
5 .0 4
1 .5
1.35
1 .2
0 .9
0 .8 5
0 .7
0.45
0.35
4.2 0
3.7 8
3.36
2.52
2.3 8
1.96
1.26
0 .9 8
A c tu a l
Og
u p ta k e
(a -x ) _
0 .8 4
1.26
1 .6 8
2 .5 2
2.66
3 .0 8
3 .7 8
4,0 6
C a lc .
Formula
u p ta k e
y * mx
0.8 5
1 .3
1.75
2 .2 1
2.66
3 .1 2
3.57
4 .0 4
m * .045
b * .39
TABLE XVIII. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN O.OOl N KCN-RINGERS
TMMEDTATFXY FOLLOWING THE SERIES TAEULATED IN TABLE XVII.__
E la p s e d
tim e i n
m in u te s
Am*t.
Thio
used
O2
E quiv.
cc/L
££
In itia l-a
10
20
30
40
50
60
70
1.875
1 .7
1 .6 5
1 .4
1 .3
1.2.
0 .8 5
A c tu a l
Og
u p ta k e
C a lc .
Og
u p ta k e
Formula
y * mx-'-b
_____________________________________________
5 .2 5
4 .7 6
4 .6 2
3 .9 2
3 .6 4
3.36
2 .3 8
.49
.6 3
1 .3 3
1 .6 1
1 .8 9
2.87
.50
.82
1 .1 5
1 .4 7
1 .8
2 .1 3
2.45
m«.0326
b .,1 7
TABLE XIX. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN MAMMALIAN RINGER SOLUTION,
288 HOURS AFTER REMOVAL FROM THE RAT AND APPROXIMATELY 130
HOURS AFTER THE LAST SERIES IN CYANIDE-RINGERS.______________
E la p se d
tim e i n
m inu tes
In itia lsa
10
20
30
40
50
60
70
Am* t .
02
Thio E quiv.
used
c c /L .
cc.
2.30
6 ,4 4 ,
2 .05
2.00
1 .8 0
1.40
1 .20
1 .00
0 .85
5 .7 4
5 .6 0
5 .0 4
3 .9 2
3 .3 6
2 .8 0
2 .3 8
A c tu a l
02
u p ta k e
(a -x )
0 .7 0
0 .8 4
1 .40
2.52
3.08
3.64
4.06
C a lc .
02
u p ta k e
0 .3 4
0.99
1 .6 4
2.2 9
2 .9 4
3 .5 9
4 .2 4
Formula
y » mx
b
m 0•06 5
b = (- 0 .3 1 )
TABLE XX. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF THE SAME LARVAE IN MAMMALIAN RINGER SOLUTION,
364 HOURS AFTER REMOVAL FROM THE RAT AND APPROXIMATELY 207
HOURS AFTER THE LAST SERIES IN CYANIDE-RINGER3.
E la p s e d
tim e i n
m in u te s
Am» t .
02
Thio E quiv.
c c /L .
used
cc.
I n i t i a l * a —I t 9..
10
20
30
40
50
60
70
80
1.7
1.7
1.35
1.25
1.05
0.8 5
0.70
0.60
A c tu a l
02
u p ta k e
(a -x )
C alc.
Op
u p tak e
Formula
y ■ mx + b
.§ .£ £ 4 .7 6
4 .7 6
3 .7 8
3 .5 0
2 .9 4
2 .3 8
1 .9 6
1 .6 8
0.28
0.2 8
1.26
1.5 4
2.10
2.66
3.08
3.36
0 .2 3
0 .7 2
1 .2 1
1 .7 0
2 .1 9
2 .6 8
3.17
3.66
m = 0.049
b = (-0 .2 6 )
TABLE XXI. SHOWING THE RESULTS OF
CONSUMPTION OF 24 £ . TAENIAEFORMIS
APPROXIMATELY. MEASUREMENTS TAKEN
SOLUTION AT APPROXIMATELY 48 HOURS
MEASUREMENTS OF OXYGEN
LARVAE WEIGHING 0 .6 GM.
IN MAMMALIAN RINGER
AFTER REMOVAL FROM THE
M X .!____________________________________________________________________________________
E la p se d
tim e i n
m in u te s
In itia l« a
10
20
30
40
50
60
Am»t.
Og
T hio E quiv.
u sed
c c /L .
cc.
1 .7 2
4 .8 1
1 .6 8
1 .4 6
1 .1 4
1 .0 7
0 .8 6
0 .6 0 2
4 .7 0
4 .0 8
3 .19
2 .9 9
2.40
1 .6 8
A c tu a l
Og
u p ta k e
(a-x)
0 .1 1
0 .7 3
1 .6 2
1 .8 2
2 .4 1
3 .1 3
C a lc .
Formula
Og
u p ta k e
y» mx-f-b
—
—________
0 .2 9
0 .8 3
1 .37
1 .9 1
2 .4 5
2 .9 9
m= 0 .0 54
b = (-0 .2 5 )
TABLE XXII. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF 24 T. TAENIAEFORMIS LARVAE WEIGHING 0 .6 GM
APPROXIMATELY. MEASUREMENTS TAKEN IN 0 .001 N KCN-RINGERS
IMMEDIATELY FOLLOWING SERIES TABULATED I N TABLE XXI.______
E lap sed
tim e i n
m inu tes
Am11.
02
Thio E quiv.
u sed
c c /L .
CC,.
In itia l-a
10
20
30
40
50
60
._
1 .7 0
1 .6
1 .4 7
1 .3 7
1 .1
0 .5 5
A c tu a l
02
u p ta k e
(a -x )
C alc.
02
u p ta k e
Formula
y s mx v-b
5*134.76
4.48
4 .11
3.8 3
3 .08
2 .1 8
0 .3 6
0 .6 4
1 .0 1
1 .2 9
2 .0 4
2 .9 4
0 .2 0
0 .67
1 .1 4
1 .6 1
2 .0 8
2.55
m = 0.047
b t. (-0 .2 7 )
TABLE X X III. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF 24 T. TAENIAEFORMIS LARVAE WEIGHING 0 .6 GM.
APPROXIMATELY. MEASUREMENTS TAKEN IN MAMMALIAN RINGER
SOLUTION AT ABOUT 72 HOURS AFTER REMOVAL FROM THE RAT. _____
E la p se d Am»t.
Og
A c tu a l C a lc .
Formula
tim e i n
T hio E quiv.
02
Og
m in u tes u sed
c c /L .
u p ta k e
u p ta k e
y*m x*b
____________ c c .____________ (a -x )________________________________
In itia l-a
10
20
50
40
50
60
2*0____ 5*£
1 .8
1 .5 5
1 .4
1 .15
0 .9
0 .6 5
5 .0 4
4 .3 4
5 .9 2
3 .2 2
2 .5 2
1 .8 2
0 .56
1 .26
1 .6 8
2 .3 8
3 .0 8
3 .78
0 .5 2
1.16
1 .8 0
2 .4 4
3 .0 8
3 .7 2
m ■*
b »
TABLE XXIV. SHOWING THE RESULTS OF MEASUREMENTS OF OXYGEN
CONSUMPTION OF 24 T. TAENIAEFORMIS LARVAE WEIGHING 0 .6 GM.
APPROXIMATELY. MEASUREMENTS TAKEN IN 0 .00 1 N KCN-RINGER
SOLUTION AT 75 HOURS FOLLOWING SHORTLY AFTER REMOVAL FROM
THE SOLUTION OF TABLE XXIII._________________________________
E la p se d Am*t.
Og
A c tu a l
C a lc .
Formula
tim e i n
Thio E qu iv.
Og
O2
m in u te s u sed
c c /L .
u p ta k e
u p ta k e
y»m x + b
____________ SSL*____________ i& zZl_______________________________
In itia l* a
10
20
30
40
50
60
2 . 0 __ 5 .6
1 .9
1.65
1 .4
1 .3
1 .1
1 .0
5 .3 2
4 .6 2
3 .9 2
3 .6 4
3 .0 8
2 .8 0
.
0 .2 8
0 .9 8
1 .6 8
1.96
2 .5 2
2.8 0
0 .5 0
0.9 8
1.46
1 .9 4
2.42
2.90
m - .048
b =. .0 2
TABLE XXV. SHOWING RATE OF METHYLENE BLUE REDUCTION IN AN
EVACUATED THUNBERG TUBE BY FIVE LARVAE OF J . TAENIAEFORMIS
USING RINGER SOLUTION CONTAINING NO GLUCOSE. DRY WEIGHT OF
THESE WORMS A FEW HOURS AFTER THIS EXPERIMENT WAS 0 .0 4 5 GM.
APPROXIMATELY._______
____
______
________________
E lap sed
tim e In
m in u tes
Observed
change I n
M ethylene
b lu e
10
20
30
60
90
120
150
180
210
240
270
300
2 .0
3 .0
3 .5
5 .5
8 .0
9 .5
1 1 .5
1 1 .5
1 4 .5
1 8 .5
1 9 .5
2 2.0
Calc* change
In
M ethylene
b lu e
2 .1 9
2 .8 5
3.51
5.4 9
7 .47
9 .4 5
1 1.43
1 3.41
1 5.39
17.37
1 9 .3 5
2 1.33
Formula
v - mx ■*- b
m “0 • 066
b -1 .5 3
TABLE XXVI. SHOWING RATI OF METHYLENE ELUE REDUCTION IN AN
EVACUATED THUNBERG TUEE BY FIVE LARVAE OF T. TAENIAEFORMIS
USING RINGER SOLUTION CONTAINING ONF PERCENT GLUCOSE. SIZE
AND WEIGHT OF THESE LARVAE WERE SIMILAR TO THE GROUP
TABULATED IN TABLE XXV.
E lap sed
tim e I n
m in u te s
Observed
change i n
M ethylene
b lue
10
20
30
60
90
120
150
180
210
240
270
300
1 .0
2 .0
1 .5
3 .5
6 .0
6 .5
8 .0
8 .0
1 1 .0
1 2 .5
1 4 .5
1 5 .5
C a lc , change
In
M ethylene
b lu e
1 .4 0
1 .8 9
2 .38
3 .8 5
5 .32
6 .7 9
8.2 6
9 .7 3
11.20
12.67
1 4.14
1 5 .6 1
Formula
y r mi + b
m - 0 .049
b * 0 .9 1
TABLE X X V I I . SHOWING AVERAGE RATE OF METHYLENE BLUE
REDUCTION IN EVACUATED THUNBERG TUBES IN RINGER SOLUTION
CONTAINING NO GLUCOSE. THREE GOUPS OF FIVE LARVAE EACH
WERE UTILIZED._________________________________________________
Observed
C a lc , change
E la p se d
change I n
in
Formula
tim e i n
M ethylene
M ethylene
m in u te s_____________ b lu e _______________ b lu e __________y - n a ^ b
10
20
30
60
90
120
150
180
210
240
270
300
1 .5
1 .5
2.2 5
4 .1 7
6 .3 3
7 .0
8 .8
9 .7
1 0 .5
1 4 .0
1 5 .3
1 6 .8
1 .4 8
2 .0
2 .5 1
4 .0 5
5.5 9
7 .1 3
8 .6 7
1 0 .2
11 .7 4
13 .2 8
14 .8 2
16.36
TABLE XXVIII. SHOWING AVERAGE RATE OF METHYLENE BLUE
REDUCTION IN EVACUATED THUNBERG TUBES IN RINGER SOLUTION
CONTAINING ONE PER CENT GLUCOSE. TWO GROUPS OF FIVE LARVAE
EACH WERE U T I L I Z E D . ___________
_____
E la p se d
tim e i n
m in u te s
10
20
30
60
90
120
150
180
210
240
270
300
Observed
change in
M ethylene
b lu e
0 .2 5
1 .0
1 .0
2 .7 5
6 .0
6 .0
7 .0
7 .0
1 0 .0
1 2 .0
1 3.25
1 4 .0
C a lc , change
in
M ethylene
blue
0 .8 4
1 .2 9
1 .7 4
3 .09
4 .4 4
5 .7 9
7 .1 4
8 .49
9 .8 4
11.19
12 .54
13.89
Formula
y * mx * b
m = 0.045
b = 0 .3 9
>4
n
22
21
20
19
I to
17
\6
15
14
13
I2
11
10
9
to
7
G
S
4
3
2
1
0
14
23
i 2
21
20
19
18
\ 7
16
15
14
13
12
I1
10
9
b
;
6
3
2
1
O
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4
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O
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-6 1 -
DISCUSSION
Through t h e l i t e r a t u r e t h e r e a r e many r e f e r e n c e s
of p a r a s i t i c worms b e in g m a in ta in e d i n v i t r o .
Much o f th e
e a r l i e r work was i n t h e n a t u r e o f a b y -p ro d u c t.
V arious
i
worms were r e p o r t e d a s having rem ained a l i v e f o r A f f e r e n t
p e r io d s o f tim e .
These somewhat chance o b s e r v a t io n s y i e l d e d
l i t t l e t h a t was t r u l y p r o d u c tiv e and s t i m u l a t i n g to p h y s io ­
lo g ic a l re search .
With th e i n v e s t i g a t i o n s o f Bunge (1883) on
a n a e ro b ic c u l t u r e o f A s c a r i s , a p e r io d was in tr o d u c e d when
s e r i o u s s tu d y and w e ll - t h o u g h t - o u t e x p e rim e n ts were a p p lie d
to p h y s i o l o g i c a l s t u d i e s o f p a r a s i t e s , as th e y had been
a p p l i e d ,a l m o s t from t h e b e g i n n i n g ,t o a n a to m ic a l and l i f e
c y c le problem s.
The i S v i t r o c u l t u r e o f p a r a s i t i c forms has so
f a r e n c o u n te re d in su rm o u n tab le d i f f i c u l t i e s .
To d a t e ,
t r i a l and e r r o r methods have proved ab o u t th e o n ly means
o f d e v i s i n g s a t i s f a c t o r y c u l t u r e media.
The c u l t u r e o f
f a c u l t a t i v e p a r a s i t e s and o f f r e e - l i v i n g s t a g e s o f o b l i g a ­
to r y p a r a s i t e s has been c o m p a ra tiv e ly e a s y .
Such s ta g e s
are a l r e a d y w e ll-a d a p te d to meet th e en vironm ent o u t s i d e
th e h o s t , so t h a t i n numerous c a s e s i t has been only a
q u e s tio n o f f i n d i n g th e most o p tim a l s i t u a t i o n .
P a r a s i t i c s t a g e s p r e s e n t an a l t o g e t h e r d i f f e r e n t
problem .
A l a r g e number o f p a r a s i t e s have been k e p t a l i v e
-6 2 -
f o r v a r y in g p e r io d s o f tim e .
Only i n e x c e p tio n a l c a s e s
has growth o r even r e g u l a t e d changes i n form been o b se rv e d .
The c o r r e c t sequence and com b in atio n o f th e optim a o f a
l a r g e number o f f a c t o r s must be a c h ie v e d .
Knowledge o f such
f a c t o r s w i l l be g a th e r e d p ie c e m e a l, m o stly from p h y s i o l o g i c a l
i n v e s t i g a t i o n s o f each f a c t o r s e p a r a t e l y .
I t i s re c o g n iz e d t h a t more a n a to m ic a l d i f f e r e n c e s
t h a n s i m i l a r i t i e s e x i s t between such d i v e r s e groups as
a c a n th o c e p h a la , nem atodes, and fla tw o rm s .
Among t h i s l a s t
g ro u p , t h e tapeworms a r e s e p a r a te d from tre m a to d e s by t h e
absen ce o f a g u t i n th e fo rm er, a f e a t u r e which a t once sug­
g e s t s profound d i f f e r e n c e s .
Absence o f an i n t e s t i n e d e t e r ­
mines t h e l o c a t i o n f o r a d u l t tapeworms, and, w ith th e ex­
c e p t i o n o f form s t h a t o c c u r i n b i l e and h e p a ti c d u c t s , th e y
a r e no rm ally found i n th e h o s t I n t e s t i n e .
T here i s , how­
e v e r , a m ajor f e a t u r e t h a t t h e s e d i v e r s e groups have i n
common,and i t i s p a r t i c u l a r l y t r u e when th e s e forms a r e p a r a ­
s i t i c i n th e same o r i n c l o s e l y r e l a t e d h o s t s .
P re c ise
a d a p t a t i o n s must be made to an environm ent which i s r a d i ­
c a l l y d i f f e r e n t and y e t which i s r i g o r o u s l y c o n t r o l l e d .
Von Brand (1938) p o in te d o u t th e g r e a t s i m i l a r i t y i n m etab o l­
ism between t h e tapeworm, M o niezia, and th e nem atode,
A sc a ris.
O b s e rv a tio n s by Weinland and von Brand (1 9 2 6 ),
and by H a rn ise h (1932) f o r F a s c i o l a ; and von Brand (1940)
f o r A cantho cephala showed t h a t th e s e forms a l s o p o s s e s s a
m etabolism s i m i l a r to A s c a r i s .
T h e r e f o r e , i t a p p e a rs t h a t
a d a p t a t i o n s o f t h e s e v a r io u s p a r a s i t e s have pursued a s im i­
l a r c o u r s e , and t h a t p h y s i o l o g i c a l convergence had o c c u rre d
to a c o n s i d e r a b l e e x t e n t .
However, a g r e a t many p a r a s i t e s can sta n d c o n s id ­
e r a b l e v a r i a t i o n s i n t h e f a c t o r s concerned w ith a p a r a s i t i c
life .
For i n s t a n c e , T a e n ia t a e n i a e f o r m i s l a r v a e , i n e x p e r i
ments j u s t r e p o r t e d , have s u rv iv e d a pH as h ig h a s 9 .3 and
a s low as 4 . 6 .
F u rth e rm o re , t h e s e worms have w ith sto o d
g ra d u a l change i n t h e pH o f th e medium from 8 .8 to 6 . 5 , and
a b r u p t change when r e t u r n e d t o t h e h ig h e r pH w ith no chance
f o r g ra d u a l a c c l i m a t i z a t i o n .
These l a r v a e a re t i s s u e p a r a ­
s i t e s , but t h e a b i l i t y (some s u rv iv e d t h e t r e a tm e n t f o r as
long a s 16 days) t o w ith s ta n d c o n s i d e r a b l e changes i n pH
may i n d i c a t e t h a t t h e worms s t u d ie d had re ach ed an age a t
which th e y would be i n f e c t i v e i n th e i n t e s t i n e o f t h e f i n a l
h o s t , where th e y might l i k e l y be s u b je c te d to c o n s id e r a b le
v a r i a t i o n i n pH.
S e v e r a l i n v e s t i g a t o r s have r e p o r t e d t h a t a pH of
a p p ro x im a te ly 7 .3 - 7 .6 appeared t o be optimum f o r i n v i t r o
m aintenan ce o f p a r a s i t i c worms, and t h i s ra n g e has been r e ­
p o r te d f o r b o th i n t e s t i n a l and t i s s u e p a r a s i t e s .
Such an
a l k a l i n e c o n d i t i o n i s more l i k e l y optimum f o r t i s s u e p a r a ­
s i t e s th a n f o r t h e o t h e r form s. F erguson (1940) r e a r e d
m e ta c e r c a r ia e to a d u l t tre m a to d e s and found
t h a t pH 6 . 2 -
-6 4 -
6 . 5 was b e s t f o r t h i s i n t e s t i n a l p a r a s i t e o f b i r d s .
Davey
(1937) r e p o r t e d t h a t sheep nem atodes o f th e u p p er sm a ll
i n t e s t i n e were a b le to w ith s ta n d pH changes o f t h e f o llo w in g
m ag n itu d e; 3 .2 - 9 . 0 and 4 .4 - 9 .0 depending upon th e
s p e c ie s and i t s norm al l o c a t i o n i n th e a lim e n ta r y c a n a l .
The q u e s t i o n o f oxygen t e n s i o n has n o t been answ ered.
A ccording to a l l r e p o r t s , th e p re se n c e o f oxygen i n a medium
i s not h arm fu l to t h e worms.
su ch oxygen.
In most c a s e s , th e y u t i l i z e
F ried h eim (1934) found t h a t t h e eg g s, p l e r o -
c e r c o i d s , and a d u l t s o f D ip h v llo b o th riu m la tu m , t h e broad
tapeworm, abso rbed oxygen; and H a rn lsc h (1933) found w ith
T ria n o p h o ru s n o d u lo s u s . a tapeworm o f f i s h , t h a t a b s o r p t i o n
was p r o p o r t i o n a l t o th e oxygen p r e s s u r e o f t h e e n v iro n m e n t,
a c o n d it i o n t h a t h o ld s f o r t h e l a r v a e o f T aenia t a e n i a e f o r m l s .
f o r oxygen c o n c e n t r a t i o n s o f s a l t s o l u t i o n s i n e q u i l i b r i u m
w ith t h e atm o sp h ere.
S u r v i v a l o f t h e s e l a r v a e i n a e r o b ic
c o n d i t i o n s was c o n s i s t e n t l y b e t t e r th a n when th e medium was
a n a e r o b i c . Even t h e l a r v a e u t i l i z e d in th e Thunberg a p p a r a tu s
i n vacuo, l i v e d f o r some h o u rs a f t e r r e s t o r a t i o n t o s a l i n e
media whose oxygen c o n te n t was i n e q u i l i b r i u m w ith th e a t ­
m osphere.
Davey (1937) found t h a t sheep nem atodes o f th e
a li m e n ta r y c a n a l d ie d w ith in 48 h o u rs i n th e com plete absence
o f oxygen.
These worms were k e p t u n d er hydrogen and Davey
d id n o t d i s c u s s t h e r e l a t i o n s h i p t o pH which m ight r e s u l t ,
b u t he concluded t h a t th e sheep i n t e s t i n a l nematodes w ith
which he worked were a e r o b ic a n im a ls .
Von Brand (1937)
measured a n a e r o b ic glyco gen consum ption i n A s c a r is un der
hydrogen.
He b e l ie v e d t h a t th e r e s p i r a t i o n o f A s c a ris was
m ainly a n a e r o b ic , but he d id n o t d i s c u s s th e p o s s i b l e e f f e c t
o f hydrogen upon pH o f th e medium.
From t h e s t a n d p o in t o f t e m p e r a tu r e , th e i n c u b a t o r
b eing u sed i n p r e li m i n a r y e x p e rim e n ts f l u c t u a t e d g r e a t l y ,
y e t t h i s f a c t o r d id n o t seem to be p a r t i c u l a r l y i m p o r ta n t.
L a t e r , a bad s w i t c h , i n an e l e c t r i c i n c u b a t o r , allow ed t h e
te m p e ra tu r e to drop t o t h a t o f th e room.
T h is had e v i d e n t l y
happened sometime b e f o r e , as th e i n c u b a to r was q u i t e c o ld ,
fforms p r e s e n t i n th e i n c u b a t o r a t t h i s tim e d id n o t seem t o
be a d v e r s e l y a f f e c t e d by t h i s low er te m p e ra tu re when r e ­
s t o r e d to normal body te m p e r a tu r e .
I t i s w e ll known t h a t
such h e lm in th s a s m i c r o f i l a r i a l i v e much lo n g e r a t i c e - c h e s t
te m p e ra tu r e s th a n a t room t e m p e r a tu r e .
I n t h e p r e s e n t ex­
p e r im e n ts , i n two c a s e s , r a t s found de8d by t h e anim al room
a t t e n d a n t and n o t im m ediately r e p o r t e d , were p la c e d i n th e
c o ld room (14°C .) where th e y were l e f t a t l e a s t 24 hours
b efo re b e in g exam ined.
L ive T a e n ia t a e n ia e f o r m ls l a r v a e
were re c o v e re d and m a in ta in e d 8 and 12 days r e s p e c t i v e l y i n
t h e i n c u b a t o r a t 3 7 .5 ° c e n t i g r a d e .
McCoy (1935) s t a t e d
t h a t p a r a s i t e s a r e r e l a t i v e l y i n s e n s i t i v e to wide v a r i a t i o n s
i n c e r t a i n e n v iro n m e n ta l f a c t o r s such a s pH, oxygen t e n s i o n ,
and te m p e r a tu r e , and t h i s sta te m e n t h o ld s f o r th e l a r v a e o f
-6 6 -
X. t a e n l a e f o r m l s a lth o u g h i t d o es n o t s o lv e th e q u e s tio n o f
o p tim a .
S u rfa c e t e n s i o n a s a p h y s i c a l f a c t o r a f f e c t i n g t h e
i n v i t r o s u r v i v a l o f p a r a s i t i c worms has n o t been p r e v io u s ly
s t u d i e d , a lth o u g h Davey (1938) c o r r e l a t e d t h e d i s t a n c e from
th e b i l e d u ct a t which d i f f e r e n t s p e c ie s o f sheep nematodes
o c c u r r e d , w ith th e c o n c e n t r a t i o n o f b i l e s a l t s t h a t each
s p e c ie s would t o l e r a t e , a n d o t h e r a u th o rs have u sed sm a ll
amounts o f b i l e s a l t s i n media f o r i n t e s t i n a l worms b ecause
o f t h e normal p re s e n c e o f such s a l t s i n t h e i n t e s t i n e .
M alkani (1 9 3 3 ), deWaele ( 1 9 3 4 ) ,and Edgar (1941) showed t h a t
b i l e s a l t s were u s e f u l i n s t i m u l a t i n g e v a g in a ti o n o f s c o l i c e s
of v a r io u s l a r v a l tapeworms.
Deffaele th o u g h t t h a t hydrogen
io n c o n c e n tr a tio n was a fundam ental f a c t o r , b u t Edgar f a i l e d
to confirm deWaele.
No d i s c u s s i o n was made o f th e p o s s i b l e
e f f e c t o f reduced s u r f a c e t e n s i o n as a f a c t o r c a u s in g evag­
in a tio n .
However, Edgar got some r e i n v a g i n a t i o n of c y s t i c e r c i
a t low er c o n c e n t r a ti o n s o f b i l e s a l t s .
I n th e p r e s e n t s tu d y ,
no d i f f e r e n c e i n s u r v i v a l was found between worms m a in ta in e d
i n norm al mammalian F in g e r s o l u t i o n to which s e v e r a l drops
o f h e x y l r e s o r c i n a l ( S . t . 37) had been added end th e c o n t r o l s .
O th e r media c o n t a i n i n g sodium t a u r o c h o l a t e were no more
f a v o r a b l e f o r v i t a l i t y o f th e l a r v a e o f X- t a e n l a e f o r m l s ,
th a n were s i m i l a r media w ith o u t t h i s b i l e s a l t .
The r e s p i r a t i o n o f t h e l a r v a e o f X* t a e n l a e f o r m i s
-6 7 -
h a s been found to be c y a n i d e - s e n s i t i v e i n p a r t , and t h i s
cy a n id e e f f e c t has been shown t o be r e v e r s i b l e .
F riedheim
(1934) showed t h a t t h e r e s p i r a t i o n o f egg s, p l e r o c e r c o i d
l a r v a e , and a d u l t s o f D ln h v llo b o th rlu m latum p o s s e s s e d a
r e s p i r a t i o n which was s e n s i t i v e to c y a n id e .
The eggs were
alm o st co m p letely c y a n i d e - s e n s i t i v e , whereas t h e a d u l t was
o n ly p a r t l y s o .
He a ls o r e p o r t e d t h a t th e a d u l t worm showed
two a b s o r p ti o n bands o f cytochrome C.
I t i s p ro b a b le t h a t
cytochrom e i s r e s p o n s i b l e f o r t h e c y a n i d e - s e n s i t i v e r e s p i r a ­
t i o n o f th e l a r v a e o f T. t a e n i a e f o r m i s . s in c e c y a n id e i s a
s p e c i f i c p o iso n f o r t h i s phase o f r e s p i r a t i o n i n a e r o b ic
a n im a ls as w e ll as p l a n t s .
Meldrum (1934) s t a t e d t h a t c y to ­
chrome "can alm o st be s t a t e d t o be a component o f ev ery
a e r o b i c c e l l , and c o n v e rs e ly t h a t i t i s a b s e n t----------- o r
alm o st a b s e n t----------- from c e l l s which l i v e a n a e r o b i c a l l y . "
S ta n n a rd , McCoy, and L a tc h fo rd (1938) found t h a t
oxygen consum ption o f T r i c h i n e l l a s p i r a l i s l a r v a e was i n ­
h i b i t e d by cy an id e and s tim u la te d by p a rap h en y len e diam ine
and carb o n monoxide.
Fenn and Cobb (1932a, b) found t h a t
f r o g and r a t muscle were a ls o s t i m u l a t e d by carbon m onoxide.
They found t h a t th e R.Q. was lo w ered to 0 .7 6 when c a r to n
monoxide re p la c e d th e n i t r o g e n r a t i o in a i r (79 p er c e n t
CO and 21 p e r c e n t O g).
F ried h eim (1934) showed t h a t no
carb o n monoxide i n h i b i t i o n o f r e s p i r a t i o n o c c u rre d w ith e g g s ,
p l e r o c e r c o i d s o r a d u l t s o f rU n h v llo b o th riu m la tu m . Adam (19552)
-6 8 -
had found a d u l t A s c a r is i n h i b i t e d by carbon monoxide.
The
i n c o n s i s t e n c i e s might be e x p la in e d upon i n d i v i d u a l and
s p e c ie s d i f f e r e n c e s a s von Brand (1958) su g g e ste d i n an
a tte m p t to r e c o n c i l e t h e a e r o b ic m etabolism of t h e sheep
nematode, o s t e r t a g i a , w ith th e a n a e r o b ic m etabolism o f A s c a r i s .
Von Brand b e l i e v e d t h a t s i n c e O s t e r t a g i a was an inco m p arab ly
s m a lle r organism th a n A s c a r i s , t i s s u e s would be t h i n n e r and
body s u r f a c e r e l a t i v e l y much l a r g e r , so t h a t oxygen d i f ­
f u s i o n , even a t low t e n s i o n s , might c o n c e iv a b ly be t h e answer
to t h e a e r o b ic r e s p i r a t i o n o f t h e sheep nematode*
E x p erim en ts w ith l a r v a e o f X« t a e n i a e f o r m i s . i n
evacuated Thunberg t u b e s , have shown t h e t t h e s e worms were
ab le t o re d u c e m ethylene b lu e slo w ly .
S in ce n e a r l y o n e - t h i r d
of t h e dry w eigh t o f t h e s e l a r v a e i s due t o glycogen ( S a l i s b u r y
and A n d e rs o n ,1938) and s in c e no a p p r e c i a b l e d i f f e r e n c e oc­
c u rre d in m eth ylene b lu e r e d u c t i o n i n mammalian R in g e rs
with and w ith o u t one p e r c e n t g lu c o s e , i t may be t h a t th e
dehydrogenase s u g g e s te d by th e m ethylene b lu e r e d u c t i o n i s
a c t i n g upon glycogen a s a s u b s t r a t e .
I t i s i n t e r e s t i n g to
note th a t von Brand (1929, 193?) observed s u c c i n i c a c id i n
th e medium a f t e r a s tu d y o f th e a n a e ro b ic d e c o m p o sitio n o f
glycogen by M oniezia.
Von Brand (1934) c o n s id e re d t h a t f a t s were e x c r e t o r y
p ro d u c ts i n A s c a ris r a t h e r th a n food r e s e r v e s . M u e l l e r (1929) d i s ­
p u ted t h i s t h e o r y on th e b a s i s o f o b s e r v a t io n s o f f a t u t i l i z a t i o n
from t h e s t o r e d l i p o i d m a t e r i a l i n t i s s u e c u l t u r e s o f th e
l a t e r a l l i n e o rg an o f A s c a r i s .
ffeinlan d and von Brand (1926)
were a b le t o d e m o n stra te m o rp h o lo g ic a lly t h e s e c r e t i o n o f
f a t p a r t i c l e s i n t o th e e x c r e t o r y system o f F a s c l o l a h e n a tic a
and Vogel and von Brand (1933) showed t h a t t h e ap p e aran c e
o f f a t p a r t i c l e s c o in c id e d w ith t h e b eg in n in g o f t h e p a r a ­
s i t i c phase o f l i f e , w h ic h ,a c c o rd in g to t h e i r c o n c e p t, would
be th e b e g in n in g o f a n a e ro b ic m etabo lism .
I t should be n o te d ,
t h a t c o n c r e t i o n s ap p e ar i n t h e e x c r e to r y system o f s t r i g e i d
m e t a c e r c a r i a e and t h a t i n c e r t a i n i n s e c t s c o n c r e t io n s s p p e a r i n t h e f a t o rg a n and t h e s e a r e p ro b a b ly e x c r e to r y i n
fu n c tio n .
Mendelsohn (1935) m a in ta in e d l a r v a e o f T aenia
t a e n i a e f o r m i s a l i v e f o r 35 days i n a medium o f balan ced s a l t
s o l u t i o n , c h ic k embryo e x t r a c t , end d i l u t e h o rs e serum by
t h e R o l le r tube method o f t i s s u e c u l t u r e .
These l a r v a e ,
however, were o n ly 15 days o ld and a p p ro x im a te ly a m i lli m e t e r
in le n g th .
Oxygen d i f f u s i o n would presum ably not be a problem
fo r larv a e of t h i s s iz e .
F u rth e rm o re , l a r v a e o f t h i s p a r a ­
s i t e a r e n o t i n f e c t i v e f o r c a t s u n t i l a f t e r a seven month
s o jo u r n i n th e r a t h o s t .
C o n seq u en tly , th e i n v i t r o
c u ltu re
o f worms which a r e a p p ro a c h in g " l a r v a l m a tu r ity " i n th e i n t e r ­
m ed iate h o s t i s a very d i f f e r e n t problem from t h a t o f young,
m inute s t a g e s .
I n p r e s e n t e x p e rim e n ts , f i v e months o ld
l a r v a e o f T a e n ia t a e n i a e f o r m i s were m a in ta in e d i n a s t e r i l e
-7 0 -
medium which c o n ta in e d o n ly a t r a c e o f g lu c o se and t h e s e
l a r v a e showed m o t i l i t y 24 days a f t e r t h e b e g in n in g of t h e
e x p e rim e n t.
I t i s i n t e r e s t i n g t o n o te i n t h i s c o n n e c tio n ,
t h a t Wardle (1937) showed t h a t glycog en c o n te n t o f M oniezia
in c r e a s e d when t h e medium c o n ta in e d g lu c o s e up t o one p e r
c e n t , b u t n o t when th e g lu c o s e c o n te n t exceeded t h i s amount.
In 1941, Green and ffa rd le got g r e a t e s t lo n g e v i t y .in v i t r o
w ith M oniezia i n a v e ry d i l u t e medium i n Tyrode.
The a d d i t i o n o f u n h e s te d c a t serum made th e
medium l e s s f a v o r a b l e f o r th e v i t a l i t y o f X» t a e n i a e f o r m i s
la rv a e i n v i t r o ,
flo e p p li, Feng, and Chu (1938) r e p o r t e d
i n j e c t i o n o f c a t serum, among o th e r t h i n g s , i n t o c y s t s o f
t h e s e same l a r v a e .
The r e s u l t s were l e s s s u c c e s s f u l th a n
w ith worms removed from t h e i r c y s t s and m a in ta in e d i n s i m i l a r
m edia.
I t seems p ro b a b le t h a t p r e s s u r e e f f e c t s caused by
th e I n j e c t i o n i n t o t h e c y s t may have a f f e c t e d t h e v i t a l i t y
o f th e s e l a r v a e , but no m ention was made o f t h i s f e a t u r e .
I n t h e p r e s e n t s t u d i e s , b a c t e r i a l c o n ta m in a tio n
has o c c u rre d i n t h e media o f c e r t a i n s e r i e s .
T h e ir p re s e n c e
has i n t e r f e r r e d w ith pH d e t e r m i n a t i o n s , a s a l r e a d y d i s c u s s e d ,
a lth o u g h i t m ight be n o ted t h a t i n t h e s e r i e s o f June 2 9 th ,
l a r v a e o f X* t a e n i a e f o r m i s caused a pH change i n s t e r i l e
medium from 8 .0 to 6 . 8 .
F u rth e rm o re , th e s e l a r v a e l i v e d f i v e
d a y s , whereas l a r v a e i n i n i t i a l l y co n tam in ated and unchanged
p o r t i o n o f t h e same s to c k medium l i v e d n in e d a y s .
N orm ally,
-7 1 -
i n t h e h a b i t a t o f t h e a d u l t ta p e w o rm ,b a c te ria a r e p r e s e n t
and t h e r e a r e no d a t a showing t h a t t h e m i c r o f l o r a
affect
tapeworms o r o t h e r m etazoa which a r e normal i n h a b i t a n t s o f
th e i n t e s t i n e .
V ario u s d i g e s t s o f t r y p t o n e , p e p to n e , u n i n f e c te d
r a t l i v e r , i n f e c t e d r a t l i v e r , and v a r io u s t i s s u e s from an
u n i n f e c te d k i t t e n have been u t i l i z e d i n a e r o b i c and a n a e ro b ic
c o n d i t i o n s and w ith p e r f u s i o n methods a s w e ll as w ith media
i n which e x c r e t o r y p ro d u c ts o f th e e x p e rim e n ta l an im als
were presum ably allow ed t o ac c u m u la te .
The one c o n c lu s io n
t h a t can be re a c h e d h e re i s t h a t th e sim p le r m edia, i n g e n e r a l ,
a r e more e f f e c t i v e i n m a in ta in in g l a r v a e o f T ae n ia t a e n i a e ­
fo rm is
i n v i t r o th a n a r e more complex m ed ia.
CONCLUSIONS
1- I n th e c o u rse o f s t u d i e s on t h e m etabolism of
T aen ia t a e n i a e f o r m i s . ev id e n c e i s p r e s e n te d f o r th e f i r s t
tim e t h a t th e r e s p i r a t i o n o f l a r v a l s t a g e s o f t h i s t a p e ­
worm a r e c y a n i d e - s e n s i t i v e i n p a r t and t h a t t h i s cyan id e
in h ib itio n is re v e rsib le .
2 - New ev id en ce i s p r e s e n t e d t h a t t h e l a r v a e o f
X. t a e n i a e f o r m i s a r e a b le to re d u ce m ethylene b lu e i n a
medium devoid o f oxygen.
The Thunberg te c h n iq u e was u t i l i z e d .
P r e lim in a r y e x p e rim e n ts r e v e a le d no d i f f e r e n c e i n m ethylene
b lu e r e d u c t i o n w ith o r w ith o u t a g lu c o se s u b s t r a t e .
-7 2 -
3- I t has been found t h a t oxygen consum ption i n
X. t a e n i a e f o r m i s l a r v a e i s dependent upon oxygen t e n s i o n
i n a e r o b i c r e s p i r a t i o n whe^ t h e medium i s i n e q u i l ib r i u m
w ith a i r .
4 - I t has been shown t h a t th e s e l a r v a e , when
p la c e d i n media o f reduced s u r f a c e t e n s i o n , l i v e d no l o n g e r
th a n c o n t r o l s .
5- W ell-d ev elo p ed l a r v a e o f X* t a e n i a e f o r m i s .
a f t e r f i v e months developm ent In th e I n te r m e d ia te h o s t ,
were m a in ta in e d a l i v e f o r 24 d a y s .
T h is c o n s t i t u t e s t h e
l o n g e s t p e r io d t h a t l a r v a e which a r e ap p ro ach in g " m a tu r ity "
I n t h e d e v e lo p m e n ta l s ta g e have been k e p t a l i v e .
6 - I t h as been found t h a t l a r v a e o f X* t a e n i a e ­
fo rm is do n o t have as good s u r v i v a l u nd er a n a e ro b ic con­
d i t i o n s s un d er a e r o b ic c o n d i t i o n s .
7 - Evidence i s p r e s e n te d t h a t t h e s e la r v a e ,w h ic h
a re t i s s u e p a r a s i t e s i n t h e r a t , a r e a b le to w ith sta n d
g ra d u a l pH changes from 9 .3 to 4 .6 and a b ru p t changes from
6 . 5 to 8 .8 .
8- I t has been shown t h a t c a t serum (u n h e ated )
was l e s s f a v o r a b le f o r s u r v i v a l o f t h e s e l a r v a e removed from
t h e i r c y s t s and m a in ta in e d Xn v i t r o th a n were the c o n t r o l s
which c o n ta in e d no serum.
9 - I t was o b serv ed t h a t la r v a e o f X» t a e n i a e f o r m i s
were a b le to s u r v iv e te m p e ra tu re changes o f c o n s i d e r a b l e
-7 3 -
ra n g e (14° to 3 7 .5 ° c e n t i g r a d e ) .
10-
A l a r g e number o f media were u s e d i n a tte m p ts
to fin d a su c c e ssfu l b asis fo r th e
v itro c u ltu re of
X. t a e n ia e f o r m is l a r v a e and a d u l t s . S u r v i v a l o f th e s e l a r v a e
was lo n g e r i n sim p le th a n i n complex media.
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