A study of cultivation methods of sulphur bacteria found in thermal waters of Yellowstone National
Park
by Henry R Kathrein
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
degree of Master of Science in Bacteriology
Montana State University
© Copyright by Henry R Kathrein (1948)
Abstract:
A chronological review of the literature on sulphur bacteria was made. The term "sulphur bacteria" has
been used to include both the non-pigmented chemosynthetic types and the pigmented photosynthetic
types. Both groups are able to oxidize hydrogen sulphide to sulphur, and store the sulphur in the form
of granules within the cells.
Various cultivation technics for these sulphur organisms were tried and it was found that sodium
sulphide is an essential metabolite for their growth and cultivation.
Attempts were made to obtain pure cultures by a series of dilutions and transfers, but with no success.
In all instances, other forms of microflora overcrowded the sulphur organisms which were being
studied. In further attempts to obtain pure cultures, a number of representative chemicals which are
known to be inhibitory to certain bacteria were used. None of these chemicals proved to be inhibitory
to the other forms present.
The organisms studied in this investigation appear to belong to the order Eubacteriales, suborder
Rhodobacteriineae, family Thiorhodaceae.
Organisms collected from thermal waters of Yellowstone National Park were used in the present
investigation. AGiCNOViLSDGElSNT
The a u th o r w ish e s t o acknow ledge th e a s s i s t a n c e g iv e n
th ro u g h o u t t h i s i n v e s t i g a t i o n by D r. F .B . C o tn e r , P r o f .
V/.G. V /a lte r and K is s K .E . H e lg re n
A STUDY OF CULTIVATION METHODS OF SJLIiTJH BACTERIA
FOUND IN THERMAL ',VATERS OF YELLOWSTONE NATIONAL PABK
by
HENRY R. KATHREIN
A THESIS
S u b m itted to th e G ra d u ate Com m ittee
in
p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts
f o r th e d e g re e o f
M a ster o f S c ie n c e i n B a c te r io lo g y
at
M ontana S t a t e C o lle g e
A pproved:
I n C harge o f M ajor Work
fl h o I I'm q n
"TT1Ysrrv! n i n e - H n rn m i+ .+ .S ft
December
1948
y/iv
2
TA3L3 OF CONTEiJTS
Page
ABSTRACT........................................................
...3
INTRODUCTION............................................. .
..4
REYIE1.',r OF LITERATURE.............................
...4
REYIEtJ , OF TECHNICS EMPLOYED............
...8
PURE CULTURE TECHNICS EMPLOYED.. .
, . 22
CLASSIFICATION OF ORGANISMS FOUND
-.2 5
DISCUSSION..................................................
..2 8
SUMMARY.........................................................
..3 2
LITERATURE CITED AND CONSULTED.. .
..3 3
EXPLANATION OF PLATE............................
..3 7
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87103
3
ABSTRACT
A c h r o n o lo g ic a l re v ie w o f th e l i t e r a t u r e on s u lp h u r
b a c t e r i a was made.
The te rm " s u lp h u r b a c t e r i a " h a s b een used
to in c lu d e b o th th e n o n -p ig m en ted chemos y n t h e t i c ty p e s and
th e pigm ented p h o to s y n th e tic ty p e s .
B oth g ro u p s a r e a b le to
o x id iz e hyd ro g en s u lp h id e to s u lp h u r , and s t o r e th e s u lp h u r
i n th e form o f g r a n u le s w ith in th e c e l l s .
V a rio u s c u l t i v a t i o n te c h n i c s f o r th e s e s u lp h u r o rg an ism s
w ere t r i e d and i t was found t h a t sodium s u lp h id e i s an e s ­
s e n t i a l m e ta b o lite f o r t h e i r g ro w th and c u l t i v a t i o n .
A tte m p ts w ere made t o o b ta in p u re c u l t u r e s by a s e r i e s
o f d i l u t i o n s and t r a n s f e r s , b u t w ith no s u c c e s s .
In a l l
i n s t a n c e s , o th e r form s o f m ic r o f lo r a overcrow ded th e s u lp h u r
o rg an ism s w hich w ere b e in g s t u d i e d .
I n f u r t h e r a tte m p ts to
o b ta in p u re c u l t u r e s , a number o f r e p r e s e n t a t i v e c h e m ic a ls
w hich a r e known t o be i n h i b i t o r y to c e r t a i n b a c t e r i a w ere
used.
None o f th e s e c h e m ic a ls p ro v ed to be i n h i b i t o r y to
th e o th e r form s p r e s e n t .
The o rg an ism s s tu d ie d i n t h i s i n v e s t i g a t i o n a p p e a r to
b elo n g to th e o r d e r E u b e c t e r i a l e s , s u b o rd e r E h o d o b a c te r lin e a e ,
f a m ily T h io rh o d a c e a e .
O rganism s c o l l e c t e d from th e rm a l w a te r s o f Y ello w sto n e
N a tio n a l P a rk w ere used i n th e p r e s e n t i n v e s t i g a t i o n .
4
A STUDY OF CULTIVATION METHODS OE SULPHUR BACTERIA
FOUND IN THERMAL WATERS OF YELLOWSTONE NATIONAL PARK
INTRODUCTION
S in c e so l i t t l e work h a s been done w ith th e s u lp h u r
o rg an ism s found i n th e rm a l w a te rs o f Y e llo w sto n e N a tio n a l
P a r k , i t was th e p u rp o se o f t h e w r i t e r to d e te rm in e a s a t i s ­
f a c t o r y method o f c u l t i v a t i n g t h e s e o rg an ism s i n o r d e r t h a t
f u r t h e r s tu d ie s m ig h t be f a c i l i t a t e d .
REVIEW OF LITERATURE
The stu d y o f s u lp h u r b a c t e r i a i s a c o m p a ra tiv e ly r e c e n t
s tu d y o f m ic ro o rg a n ism s.
A lth o u g h c a s u a l o b s e r v a tio n s o f
co m p o site s u lp h u r b a c t e r i a i n h o t s p r in g s w ere made a s e a r l y
a s i 860, s t u d i e s a s to t h e i r i s o l a t i o n and p u re c u l t u r e ,
m orphology and p h y s io lo g y w ere n o t made u n t i l s e v e r a l y e a rs
la te r.
Cram er (1870) was th e f i r s t to s u g g e s t t h a t g r a n u le s
i n B e g g ia to a « a gen u s o f th e s u lp h u r b a c t e r i a , c o n s is te d o f
s u lp h u r .
From i n v e s t i g a t i o n s c a r r i e d o u t i n 1869-71 on th e
v e g e ta t io n o f th e Y e llo w sto n e H ot S p r in g s , H a rsh b a rg e r (1397)
r e p o r te d th e p re s e n c e o f b a c t e r i a a b le to d e p o s it s u lp h u r as
g r a n u le s w ith in t h e i r c e l l s .
Cohn (1875) th e n p o s t u l a t e d th e
th e o r y t h a t th e b e g g ia to a and th e p u r p le b a c t e r i a p roduce
hydrogen s u lp h id e by r e d u c tio n o f s u l p h a t e s .
Weed (1889) in
h i s p a p e r e n t i t l e d "The V e g e ta tio n o f H ot S p rin g s " shows t h a t
" t r a v e r t i n e " i s th e r e s u l t o f s u lp h u r d e p o s itio n by th e b eg ­
g ia to a .
T h is w ork was s u b s t a n t i a t e d by D av is (1 8 9 7 ).
H is
5
o b s e r v a tio n s w ere t h a t ’’t r a v e r t i n e ” and ’’f e l t ” , a c l o s e l y
woven m ass o f f ila m e n to u s b a c t e r i a i n w hich c r y s t a l s o f c a l ­
cium c a rb o n a te w ere im bedded, w ere r e s p o n s i b le f o r many o f
th e c o lo re d d e p o s its i n th e p a r k .
C o n tro v e rsy a s t o th e
o r i g i n o f o rg an ism s i n s u lp h u r s p r in g s was th e r e s u l t o f
i n v e s t i g a t i o n s c a r r i e d o u t by S e t c h e l l (1 9 0 3 ).
E is co n ­
t e n t i o n was t h a t no o rg a n ism s w ere fo u n d i n s t r i c t l y th e rm a l
w a te rs n o r i n s p r in g s w hich w ere re p u te d to have a d e c id e d
a c id r e a c t i o n .
Engelm ann (1887) f i r s t p o s tu l a te d t h a t p u r p le and g re e n
s u lp h u r b a c t e r i a b elo n g ed to th e p h o to s y n th e tic g ro u p o f
o rg a n ism s.
T h is th e o r y was s t r o n g l y opposed by W inogradsky
(1888) who p ro p o sed th e th e o r y o f c h e m o sy n th e tic m e ta b o lism .
I n th e s e p r o c e s s e s , th e en e rg y su p p ly o f th e o rg a n ism i s n o t
f u r n is h e d by d e c o m p o sitio n o f o r g a n ic m a t t e r , b u t by th e
o x id a tio n o f in o r g a n ic s u b s ta n c e s .
I n th e s e p r o c e s s e s , a l s o ,
h y drogen s u lp h id e i s o x id iz e d by th e o rg an ism to s u lp h u r ic
a c id .
M o lisc h (1907) p u b lis h e d h i s m onograph on th e p u r p le
b a c t e r i a in w hich he co n clu d ed t h a t p u r p le b a c t e r i a a s s im i­
l a t e o r g a n ic compounds i n th e l i g h t .
T h is was h i s a tte m p t
to d e f e a t th e th e o r y o f an a u to tr o p h ic mode o f l i f e f o r
th e s e o rg a n ism s, a s o u t l i n e d by p re v io u s i n v e s t i g a t o r s .
Such
a view was i n d i r e c t s u p p o r t o f th e w ork o f Nadson (1 9 0 3)
who s t a t e d t h a t h y d ro g en s u lp h id e i s n o t r e q u ir e d f o r n u t r i ­
t i o n , and s u lp h u r i s n o t a c c u m u la te d .
B uder (1 9 1 9 ), i n
6
d is c u s s in g th e v a lu e o f th e v a r io u s t h e o r i e s p r e s e n te d up
u n t i l t h i s tim e , was i n c l i n e d to b e l ie v e t h a t th e m e ta b o lism
o f th e p u r p le b a c t e r i a sh o u ld be c o n s id e re d a s a c o m b in atio n
o f p h o to s y n th e tic and chemos y n t h e t i c modes o f l i f e , in d e ­
p e n d e n t o f each o t h e r , b u t p r o v id in g th e o rg an ism s w ith th e
f a c u l t y t o l i v e and t h r i v e u n d er d iv e r g e n t c o n d i tio n s .
T h is
id e a i s c a l l e d " o n ly a w e ll founded assu m p tio n " b u t even a t
th e p r e s e n t tim e , we h ave come no f u r t h e r i n o u r know ledge
o f th e s e m e ta b o lic p r o c e s s e s .
Warming (1875) and L a n k e s te r (1 8 7 6 ), i n t h e i r e a r l y
i n v e s t i g a t i o n s , drew th e c o n c lu s io n t h a t a l l th e v a r io u s form s
and sh a p es o f c o lo re d o rg an ism s w ith d r o p l e t s i n s i d e th e c e l l s
r e p r e s e n te d d i f f e r e n t d e v e lo p m e n ta l s ta g e s o r " p h a s e s o f
grow th" o f one s p e c ie s .
T h is id e a was a tta c k e d by Cohn (1875)
who h e ld to th e m o n o m o rp h istic v ie w p o in t, a s d id W inogradsky
(1 8 8 7 ).
Such a v ie w p o in t s t r e s s e d th e f a c t t h a t d i s t i n c t
v a r i a t i o n s w ere c h a r a c t e r i s t i c o f d i f f e r e n t s p e c i e s .
On t h i s
b a s i s , W inogradsky e s t a b l i s h e d an e l a b o r a t e sy stem o f c l a s s ­
i f i c a t i o n o f th e s u lp h u r b a c t e r i a b ased upon th e sh ap e and
s iz e o f th e c e l l s , a s w e ll a s upon t h e i r mode o f c o lo n y f o r ­
m a tio n .
T h is sy stem h a s been r e t a i n e d —w ith o n ly m inor
m o d if ic a ti o n s — t o th e p r e s e n t d a y .
Van K ie l (1 9 3 0 ), th ro u g h
e x te n s iv e i n v e s t i g a t i o n , h a s co n clu d ed t h a t v a r i a t i o n s a s to
s i z e , s h a p e , and grow th a r e o f t e n e n c o u n te re d w ith in a p u re
c u l t u r e , and a r e th e r e s u l t o f e n v iro n m e n ta l e f f e c t s such a s
7
hydrogen s u lp h id e c o n c e n tr a t io n , pH o f th e medium, age o f
th e c u l t u r e , and p r e s e n c e o f f r e e oxygen.
F r o b is h e r (1944) p la c e s a l l th e s u lp h u r b a c t e r i a i n th e
o rd e r T h lo b a c te r ia le s .
C r i t e r i a f o r f u r t h e r s u b d iv is io n were
th e p re s e n c e o f p h o to s y n th e tic p ig m e n ts and th e p r e s e n c e o f
f r e e s u lp h u r a s g r a n u le s w ith in th e c e l l w a l l s .
Those o rg a n ­
ism s w hich p o s s e s s e d p h o to s y n th e tic p ig m e n ts and s t o r e s u lp h u r
w ith in t h e i r c e l l w a lls w ere c l a s s i f i e d u n d er th e fa m ily T h lo rh o d a c e a e .
Those o rg a n ism s w hich p o s s e s s e d p h o to s y n th e tic
p ig m e n ts and d id n o t s t o r e s u lp h u r w ith in t h e i r c e l l w a lls
w ere c l a s s i f i e d u n d er th e f a m ily A th io rh o d a c e a e .
Those o rg a n ­
ism s w hich p o s s e s s e d no p h o to s y n th e tic p ig m e n ts b u t s t o r e d
s u lp h u r w ith in t h e i r c e l l w a lls w ere d iv id e d i n t o th e f i l a ­
m entous o rg an ism s u n d e r th e fa m ily B e g g ia to a o e a e 1 and th e non­
f ila m e n t ous o rg an ism s u n d e r th e f a m ily A c h ro m a tla c e a e .
The
s u lp h u r b a c t e r i a have been r e c l a s s i f i e d , however, i n th e s i x t h
e d i t i o n o f S e rg e y ’ s M anual.
I n t h i s c l a s s i f i c a t i o n , th e
s u lp h u r b a c t e r i a w hich re se m b le t r u e b a c t e r i a i n m orphology
have been p la c e d u n d er O rder I , E u b a c t e r i a l e s . S u b o rd e r I I I ,
R h o d o b a c te riln e a e . and th e s u lp h u r b a c t e r i a w hich re se m b le
a lg a e i n m orphology b u t do n o t p o s s e s s p ig m e n ts u n d er O rder
I I I , C h la m y d o b a o te rla le s .
E llis
(1932) i n h i s i n v e s t i g a t i o n s on th e s u lp h u r b a c t e r i a
was th e f i r s t to u se a ch e m ic a l compound t o show th e e x is te n c e
o f s u lp h u r g r a n u le s by c o l o r i n d i c a t i o n .
When a smear o f th e
8
s u lp h u r c o n ta in in g o rg an ism s was t r e a t e d w ith a c o n c e n tr a te d
s o l u t i o n o f sodium n i t r o p r u s s i d e
(I^FelTO (CN )5 ) , th e r i n g s o f
s u lp h u r assumed a b lo o d r e d c o l o r .
T h is p ro c e d u re in d ic a t e d
th e p re s e n c e o f s u lp h u r g r a n u le s ; how ever, i t was o f no v a lu e
i n d e te rm in in g c e l l m orphology s in c e i t f a i l e d to show th e
c e ll o u tlin e .
Howard (1948) i n a stu d y o f s u lp h u r b a c t e r i a ,
dev elo p ed a d i f f e r e n t i a l s t a i n i n g te c h n ic i n w hich m a la c h ite
g re e n o r m eth y len e b lu e was used to s t a i n th e c e l l , and a f t e r
m o rd a n tin g w ith ta n n ic a c i d , sodium n i t r o p r u s s i d e was added
to s t a i n th e s u lp h u r g r a n u le s .
e s v ie ;; of tec h n ic s e l t io y sd
One o f th e g r e a t e s t d i f f i c u l t i e s e n c o u n te re d by i n v e s t i ­
g a t o r s i n th e f i e l d o f s u lp h u r b a c t e r i a h a s b een th e d e v e lo p . m ent o f m edia and te c h n i c s s u i t a b l e f o r th e i s o l a t i o n and
c u l t i v a t i o n o f p u re c u l t u r e s .
L o lis c h (190?) em ployed a s o l i d
medium c o n ta in in g r i v e r w a te r , p e p to n e , d e x t r i n , and a g a r .
However, t h i s medium c o n ta in e d an e x c e s s o f o r g a n ic m a t e r i a l
w hich would n o t p e rm it e x a c t s t u d i e s o f a u to tr o p h ic fo rm s.
K ie l (1912) employed a s t r i c t l y in o r g a n ic medium con­
t a i n i n g th e f o llo w in g c o n s t i t u e n t s :
CaH2 (CO3 )2----------------0.34%
Ca3 (PO4 ) 2 ---------------- 0 .0 2 #
LgE2 (CO3 )2 ----------------0 .2 7 #
K C l------ ------------------- 0 .0 1 #
CaSO4------------- ----------- 0 .3 1 #
K2S--------------------------- 0 .0 1 #
L pSO .
FeS--------------------------- 0 .0 1 #
CaS---------------------------0 .0 1 #
9
A sm a ll amount o f ammonium s u lp h a te was ad d ed ; a ls o oxygen,
hydrogen s u lp h id e and ca rb o n d io x id e w ere in tr o d u c e d .
Van N ie l (1930) o b ta in e d v e ry s a t i s f a c t o r y r e s u l t s w ith
a medium o f th e f o llo w in g c o m p o sitio n :
NH4C l - ........................ ........................... 0
. 1#
K2HPO4-------------------------------------- 0
LIgGl2- - ———————————
. 02#
NaHCO3--------------------------------------- 0
. 1#
Na2S ^ H 2O------------- -------------- 0
. 1#
The medium was a d ju s te d t o a pH o f 8 .0 - 3 .5 by th e a d d i tio n
o f s t e r i l e Na2C ^ o r H^PO^.
The u se o f -Na2S
by van
N ie l a s a so u rc e o f h y d ro g en s u lp h id e was b ased on e x p e r i­
m e n ta l e v id e n c e o b ta in e d w hich i n d ic a t e d t h a t many s u lp h u r
b a c t e r i a w ere c a p a b le o f o x id iz in g v a r io u s in o r g a n ic s u lp h u r
compounds t o s u lp h a te w ith th e s im u lta n e o u s p h o to r e d u c tio n
o f ca rb o n d io x id e .
Van N ie l found t h a t c u l t u r e s o f s u lp h u r b a c t e r i a w hich
dev elo p ed u n d er n a t u r a l d a y l i g h t c o n d itio n s i n th e l a b o r a to r y
i n t h r e e to f o u r w eeks showed th e same g ro w th i n f o u r to f i v e
d ay s when th e y w ere illu m in a te d c o n tin u o u s ly by an o r d in a r y
e l e c t r i c l i g h t b u lb o f 25-50 w a t t s , p la c e d a t a d i s t a n c e o f
20-30 cm from th e c u l t u r e s .
D uring i n v e s t i g a t i o n s c a r r i e d o u t by Howard (1948) u s in g
b o th K i e l ’ s in o r g a n ic medium and v an N i e l ’ s in o r g a n ic medium,
i t was found t h a t K i e l ’ s medium had l i t t l e
s e l e c t i v e v a l u e 'f o r
10
s u lp h u r b a c t e r i a s in c e a lg a e and diatom s a ls o s u r v iv e d .
How­
e v e r , van K i e l ’ s medium seemed to be s p e c i f i c f o r b a c t e r i a l
g ro w th .
Few er number o f a lg a e and d ia to m s w ere o b se rv e d i n
t h i s ty p e o f medium.
I n n e i t h e r medium was he a b le t o c a r r y
a c u l t u r e beyond th e second t r a n s f e r , i n d i c a t i n g t h a t some
e s s e n t i a l m e ta b o lite was m is s in g .
Ten sam p les used f o r th e p r e s e n t stu d y o f c u l t i v a t i o n
te c h n ic s o f s u lp h u r b a c t e r i a w ere c o l l e c t e d i n Y ello w sto n e
N a tio n a l l a r k from th e rm a l w a te rs h a v in g a d i s t i n c t o d o r o f
hydrogen s u lp h id e .
D ata c o n c e rn in g th e s o u r c e , te m p e ra tu re
a t tim e o f c o l l e c t i o n , and pH o f sam ples a re l i s t e d i n T ab le
I.
The pH m easurem ents w ere made by means o f th e Beckman pH
m e te r upon r e t u r n i n g t o th e l a b o r a t o r y .
One m i l i l i t e r o f each sam ple was in o c u la te d i n t o 1 .5 by
15 cm tu b e s c o n ta in in g 10 ml p o r t i o n s o f van K i e l ’ s medium.
I n an a tte m p t to s im u la te n a t u r a l e n v iro n m e n ta l c o n d i tio n s ,
sam ples 1 , 2 ,
3 , and 6 , h a v in g a pH ra n g e o f 8 .1 - 8 .4 w ere
in o c u la te d i n t o t h i s medium a d ju s te d to pH 8.3«
Sam ples 4 ,
5, 9» and 10, h av in g a pH ra n g e o f 7 . 2 - 7 .8 w ere in o c u la te d
i n t o t h i s same b a s ic medium a d ju s te d to pH 7 . 5 , and sam p les
7 and 8 , h a v in g a pH ra n g e o f 6 . 2 - 6 . 4 , w ere in o c u la te d i n t o
th e b a s ic medium a d ju s te d to pH 6 . 3 .
P h o s p h o ric a c id was
used t o a d j u s t th e pH l e v e l s .
P rim a ry in o c u l a t i o n s w ere made in d u p l i c a t e and w i l l be
la b e le d h e r e a f t e r T r a n s f e r No. I .
B oth s e t s o f c u l t u r e s
11
TABLE
I
Sam ples C o lle c te d From T herm al W aters o f
Y ello w sto n e N a tio n a l P a rk Showing T em p e ra tu res
and pH o f th e Sam ples f o r th e D i f f e r e n t L o c a tio n s
Sample
No.
L o c a tio n
I.
M irro r F o o l, B i s c u i t B a sin - S o u th
edge o f p o o l.
60
8 .5
2.
M ir r o r P o o l, B i s c u i t B a sin - E a s t
edge o f p o o l.
69
8 .5
3.
M ir r o r P o o l, B i s c u i t B a sin - N o rth
edge o f p o o l. D ra in a g e a r e a .
29
8 .1
4.
M irro r P o o l, B i s c u i t B a sin - W est
edge o f p o o l. D ra in ag e a r e a .
12
7 .8
5.
M irro r P o o l, B i s c u i t B a s in - N o rth
edge o f p o o l.
56
7 .7
6.
C a u lif lo w e r P o o l, B i s c u i t B a sin - E a st
edge o f p o o l. D ra in a g e a r e a .
29
8 .1
7.
T e rra c e Road, Mammoth T e r r a c e s , Forma­
t i o n Loop R oad. S m all p o o l around
H2S b u b b lin g h o le .
29
6 .4
8.
T e rra c e Road, Mammoth T e r r a c e s , Form a­
t i o n Loop R oad, S m all p o o l aro u n d
H2S b u b b lin g h o le .
39
6 .2
9.
F o rm a tio n Loop R oad, Mammoth T e r r a c e s
Orange Mound S p rin g
55
7 .2
O
H
F o rm a tio n Loop Road, Mammoth T e rra c e s
O range Mound S p rin g
58
7 .3
Temp. C
PH
12
r e c e iv e d a n i n i t i a l amount o f Na2S ^ H 2O (0 .0 1 g j .
One s e t
o f c u l t u r e s r e c e iv e d no a d d i t i o n a l su p p ly o f sodium s u lp h id e ;
how ever, th e o th e r s e t o f c u l t u r e s r e c e iv e d su p p le m e n ta l
am ounts o f 0 .0 1 g Ka2S*9E20 a t t h r e e day i n t e r v a l s .
A ll
c u l t u r e s w ere in c u b a te d a e r o b i c a l l y in a c a b in e t illu m in a te d
by two 25 w a tt b u lb s and m a in ta in e d a t a te m p e ra tu re o f 40 C.
Sam ples I , 5, and 7 w ere s e l e c t e d f o r f u r t h e r s tu d y b ec au se
th e y r e p r e s e n te d th e t h r e e d i f f e r e n t pH ra n g e s used i n th e s e
i n v e s t i g a t i o n s , nam ely 8 .3 , 7 .5 , and 6 .3 .
C ounts o f th e
number o f o rg an ism s p r e s e n t w ere made on th e s e sam ples a t th e
tim e o f th e i n i t i a l i n o c u l a t i o n , and t h e r e a f t e r a t 2 day i n t e r ­
v a ls .
A' Breed m ilk p i p e t t e t h a t d e l i v e r s 0 .0 1 ml was employed
and th e c o u n ts w ere made by s p r e a d in g 0 .0 1 ml p o r t i o n s o f th e
sample o v e r an a re a o f one sq u a re c e n tim e te r on a c le a n g l a s s
s lid e .
The sm ears were th e n f i x e d by h e a t , s ta in e d w ith
c a r b o l f u c h s in , w ashed, d r ie d and o b se rv e d u n d er th e o i l
im m ersion o b j e c t i v e .
C o u n ts w ere made o n ly o f t y p i c a l s u lp h u r
o rg an ism s o f one m o rp h o lo g ic a l ty p e , w hich had b een e s t a b l i s h e d
a s s u lp h u r b a c t e r i a by m eans o f th e sodium n i t r o p r u s s i d e s t a i n
dev elo p ed by Howard (1 9 4 8 ).
Twenty f i e l d s w ere co u n ted and
th e a v e ra g e o f th e number o f su ch o rg an ism s p e r f i e l d was
m u l t i p l i e d by th e m ic ro s c o p ic f a c t o r (5 6 5 ,0 0 0 ) t h a t had been
d e te rm in e d p r e v io u s l y .
A c o m p ila tio n o f c o u n ts and th e lo g a r ith m s o f th e c o u n ts
on T r a n s f e r No. I a r e shown i n T a b le I I .
G raphs show ing th e
13
TABLE I I
T r a n s f e r No. I C o u n ts o f S u lp h u r
O rganism s Showing E f f e c t o f N a g S * ? ! ^ on grow th
C u ltu r e s r e c e iv in g Na2S • 9H2O a t 3 day i n t e r v a l s
Sample I
Sample 5
D ate
Number
x 10;
Log.
Number
x 10%
Log.
9 /2 7
9 /2 9
1 0 /2
1 0 /4
1 0 /6
1 0 /9
1 0 /1 1
10/13
10/15
1 0 /1 7
932
1 ,4 6 9
2 ,4 2 9
4 ,3 5 0
6 ,0 4 5
8 ,4 1 8
11,582
1 1 ,3 0 5 ,
8 ,9 2 7
6 ,1 5 2
5 .9
6 .2
6 .3
6 .6
6 .7
6 .9
7 .1
7 .1
6 .9
6 .8
621
1 ,1 8 6
2 ,9 9 4
4 ,4 1 5
8 ,7 5 7
1 2 ,6 1 2
1 3 ,9 4 4
2 2 ,7 6 9
1 7 ,6 7 9
1 0 ,6 7 8
5 .8
6 .1
6 .5
6 .6
6 .9
7 .1
7 .2
7 .4
7 .2
7 .0
Sample 7
Number
x 103
297
508
1 ,1 4 3
1 ,1 8 6
1,2 9 9
1 ,2 9 9
2 ,8 8 1
5,7 0 6
5,7 1 9
4 ,5 7 6
Log.
5 .5
5 .7
6 .1
6 .1
6 .1
6 .1
6 .5
6 .8
6 .8
6 .7
C u ltu r e s r e c e iv in g Na2S ^ H 2O o n ly a t s t a r t
9 /2 7
9 /2 9
1 0/2
1 0 /4
1 0 /6
1 0 /9
1 0/11
1 0/13
1 0/15
1 0/17
706
1 ,2 4 3
1 ,1 2 9
1 ,0 1 5
791
678
452
282
169
113
5 .8
6 .1
6 .1
6 .0
5 .9
5 .8
5 .7
5 .4
5 .2
5 .1
565
1 ,3 5 6
1 ,4 1 2
1 ,4 6 9
1 ,1 7 3
960
565
339
282
226
5 .8
6 .1
6 .1
6 .2
6 .1
6 .0
5 .8
5 .5
5 .5
5 .4
169
734
621
678
621
508
339
226
113
56
5 .2
5 .9
5 .8
5 .8
5 .7
5 .7
5 .5
5 .4
5 .1
4 .8
14
F ig u re I . T r a n s f e r No. I E f f e c t o f Na2S / 9H20 on grow th
o f s u lp h u r b a c t e r i a i n an in o r g a n ic medium. Red i n d i ­
c a te s c u l t u r e s r e c e iv in g N82S a t t h r e e day i n t e r v a l s .
B lue i n d i c a t e s c u l t u r e s r e c e iv in g IIa2S o n ly a t s t a r t .
e f f e c t o f Na2S»9H20 on th e grow th o f s u lp h u r b a c t e r i a a re
in c lu d e d i n F ig u re I .
F ig u re I (a) r e p r e s e n t s sam ple No. I
c o l l e c t e d from M irro r P o o l and h a v in g a pH o f 8 . 4 .
F ig u re I
(b) r e p r e s e n t s sam ple No. 5 , a ls o c o l l e c t e d a t M irro r P o o l
and h a v in g a pH o f 7 .7 ; and F ig u re I (c) r e p r e s e n t s sam ple
No. 7 c o l l e c t e d a t Manmoth t e r r a c e s , h a v in g a pH o f 6 . 4 .
Red l i n e s i n d i c a t e c u l t u r e s w hich r e c e iv e d Na2S»9H20 a t th r e e
day i n t e r v a l s ; b lu e l i n e s i n d i c a t e c u l t u r e s w hich r e c e iv e d
o n ly an i n i t i a l su p p ly o f Na2S»9H20.
As can be se en from th e c o u n ts i n T ab le I I and a ls o from
th e g r a p h ic a l p r e s e n t a t i o n o f t h e s e c o u n ts i n F ig u re I , b o th
s e t s o f c u l t u r e s e x h i b i t a p p ro x im a te ly th e same amount o f
grow th d u rin g th e f i r s t few d a y s .
However, a f t e r a p e r io d
o f t h r e e to f o u r days a f t e r i n o c u l a t i o n , th o s e c u l t u r e s w hich
r e c e iv e d su p p le m e n ta l in c re m e n ts o f Na2S*9H20 show a d e f i n i t e
in c r e a s e i n num ber, w h ereas th o s e c u l t u r e s w hich r e c e iv e d no
a d d i t i o n a l am ounts o f
e x h i b i t a s t a t i o n a r y p h ase o r
a p h ase o f s l i g h t d e c re a s e i n num ber.
T h is d i f f e r e n c e i s
f u r t h e r a c c e n tu a te d a f t e r a grow th p e r io d o f 16 d a y s .
Those
c u l t u r e s r e c e iv in g s u p p le m e n ta l am ounts o f sodium s u lp h id e
show a 10-15 f o l d in c r e a s e i n number o v e r th o s e i n th e o r i g i n a l
in o cu lu m , w h ereas i n th o s e c u l t u r e s r e c e iv in g no s u p p le m e n ta l
sodium s u lp h id e , th e num bers w ere red u ce d fro m two to t h r e e
.■/r
tim e s th e o r i g i n a l number o f o rg a n ism s.
f r
,
T hese d a ta i n d i c a t e , ,
t h a t th e a d d i tio n o f sodium s u lp h id e h a s a d e c id e d s ti m u la to r y
16
e f f e c t upon th e g ro w th o f s u lp h u r o rg a n ism s.
Sam ples Mo. I and 5 te n d to e x h i b i t th e same g e n e r a l
g row th p a t t e r n .
How ever, i t w i l l be n o tic e d t h a t sam ple
No. 7 d id n o t grow a s w e ll, n o r d id i t r e a c h th e maximum grow th
shown by sam ples I and 5 * a lth o u g h th e c e l l s re se m b le th o s e
i n I and 5 m o r p h o lo g ic a lly .
D e s p ite t h i s f a c t , a s h a rp d i s ­
t i n c t i o n i s shown betw een th e c u l t u r e s r e c e iv in g su p p le m e n ta l
am ounts o f sodium s u lp h id e and th e c u l t u r e s r e c e iv in g no
a d d i t i o n a l am ounts o f sodium s u lp h id e .
A f te r 14 d ay s o f in c u b a ti o n , th e t e n sam p les from T r a n s f e r
No. I w ere in o c u la te d i n t o 10 ml p o r t i o n s o f f r e s h medium, u s ­
in g an inoculum o f one ml i n ea ch c a s e .
A gain i n o c u l a t i o n s w ere
made i n d u p l i c a t e , one s e t r e c e i v i n g o n ly an i n i t i a l su p p ly o f
Na2S e9H2O and th e o th e r s e t r e c e iv in g su p p le m e n ta l in c re m e n ts
o f Na2S e9H2O a t t h r e e day i n t e r v a l s in a d d i t i o n to th e i n i t i a l
s u p p ly .
The same te c h n i c s o f i n o c u l a t i o n and c o u n tin g w ere
c a r r i e d o u t on T r a n s f e r No. 2 a s f o r T r a n s f e r No. I .
C om pila­
t i o n o f c o u n ts and th e lo g a r ith m s o f th e c o u n ts on T r a n s f e r
No. 2 a r e shown i n T ab le I I I .
The e f f e c t o f Na2S e9H2O on th e
grow th o f s u lp h u r b a c t e r i a i s shown i n F ig u re 2 .
F ig u re 2 (a)
r e p r e s e n t s c u l t u r e No. I , F ig u r e 2 ( b ) , c u l t u r e No. 5» a&d
F ig u r e 2 ( c ) , c u l t u r e No. 7»
A f te r an in c u b a tio n p e r io d o f 14 d a y s , th e sam ples from
T r a n s f e r No. 2 w ere r e - i n o c u l a t e d i n t o 10 ml p o r t i o n s o f f r e s h
medium, a g a in u s in g an in o cu lu m o f I m l.
T r a n s f e r No. 3 was
17
TABLE I I I
T r a n s f e r No. 2 C ounts o f S u lp h u r
O rganism s Showing E f f e c t o f
on Growth
C u ltu r e s r e c e iv in g IIa2S«9H20 a t 3 day i n t e r v a l s
Sample I
D ate
1 0 /1 1
10/13
10/15
1 0 /1 8
1 0/20
1 0/22
10/25
1 0 /2 7
1 0 /2 9
n /i
1 1 /3
Number
x 103
113
395
1 ,6 9 5
2 ,4 2 9
3,616
5,189
5 ,2 5 4
5,3 6 7
4 ,6 8 9
4,5 2 0
4 ,0 6 8
Sample 5
Log.
5 .1
5 .6
6.2
6 .4
6 .6
6 .7
6 .7
6 .7
6 .7
6 .6
6.6
Number
x 10 3
254
960
1 ,7 5 1
2 ,6 5 5
4 ,4 6 3
6 ,5 5 4
6 ,4 9 7
6 ,0 4 5
5 ,3 6 7
5 ,2 5 4
4 ,9 7 2
Log.
5 .4
5.9
6 .2
6 .4
6 .6
6 .8
6 .8
6 .8
6 .7
6 .7
6 .7
Sample 7
Number
x 103
56
169
565
1 ,1 8 6
2 ,0 3 4
3 ,1 0 7
3 ,1 6 4
3 ,0 5 1
2 ,9 3 8
2 ,8 2 5
2 ,4 2 9
Log.
4 .7
5 .2
5 .7
6 .1
6 .3
6 .5
6 .5
6 .5
6 .5
6 .4
6 .4
C u ltu r e s r e c e iv in g Na23*9H20 o n ly a t s t a r t
1 0 /1 1
10/13
10/15
1 0 /1 8
1 0 /2 0
10/22
10/25
1 0 /2 7
1 0 /2 9
1 1 /1
11 /3
141
508
678
282
226
226
169
56
56
56
28
5 .1
5 .7
5 .8
5 .4
5 .4
5 .4
5 .2
4 .6
4 .6
4*6
4 .4
282
524
734
339
339
282
282
169
113
56
56
5 .5
5 .7
5 .9
5 .5
5 .5
5 .4
5 .4
5 .2
5 .1
4 .6
4*6
28
232
226
56
28
4 .5
5 .5
5 .4
4 .7
4 .5
■
-
—
1—
—
—
—
—
—
—
—
18
7
6
5
4
a.
7
6
5
4
7
6
5
4
2
4
6
8
10
12
14
16
18
20
22
24
TIMifi IN DAYS
T r a n s f e r No. 2 E f f e c t o f Na^S.SHgO on grow th
b a c t e r i a in an in o r g a n ic medium. Red i n d i c a t e s
r e c e iv in g NagS a t t h r e e day i n t e r v a l s . Blue
c u l t u r e s r e c e iv in g Na2S o n ly a t th e s t a r t .
19
a ls o made i n d u p l i c a t e , one s e t o f c u l t u r e s r e c e i v i n g o n ly
an i n i t i a l su p p ly o f Na2S ^ H 2O, th e o th e r s e t r e c e iv in g su p ­
p le m e n ta l am ounts o f Na2S ^ H 2O a t t h r e e day i n t e r v a l s In
a d d i tio n to th e i n i t i a l s u p p ly .
S im il a r d a ta h ave been r e ­
corded f o r T r a n s f e r No. 3 a s f o r T r a n s f e r No. I and 2 .
C ounts
o f o rg a n ism s a r e com piled i n T ab le IV, and g ra p h s show ing th e
e f f e c t o f NagS'SH^O on th e g ro w th o f s u lp h u r o rg a n is m s ,
c u l t u r e s I , 5, and 7 , a r e shown u n d er F ig u re 3 ( a ) ,
( b ) , and
(c) r e s p e c t i v e l y .
The g e n e r a l p a t t e r n s o f g ro w th f o r T r a n s f e r No. 2 and 3
a r e v e ry s im ila r to th o s e o b se rv e d i n T r a n s f e r No. I .
I t w ill
be n o te d t h a t maximum grow th i n T r a n s f e r s No. 2 and 3
is
below t h a t e x h ib ite d i n T r a n s f e r No. I .
S in c e o th e r fo rm s o f
m ic r o f lo r a w ere p r e s e n t i n th e s e t r a n s f e r s , i t was f e l t t h a t
th e y m ig h t have some m in o r i n h i b i t o r y e f f e c t upon th e grow th
o f th e s u lp h u r o rg a n ism s.
I t was n o tic e d i n a l l t h r e e t r a n s f e r s t h a t sodium s u l ­
p h id e had a d e c id e d e f f e c t on th e g ro w th o f s u lp h u r o rg a n ism s.
Those c u l t u r e s r e c e iv in g s u p p le m e n ta l in c re m e n ts o f Na2S ^ H 2O
showed a d e f i n i t e in c r e a s e i n num bers up to a c e r t a i n s t a g e .
T r a n s f e r s co u ld be made i n t o f r e s h m edia a t th e p e a k o f grow th
and a new c y c le o f grow th w ould be e s t a b l i s h e d .
However, th o s e
c u l t u r e s r e c e iv in g o n ly an i n i t i a l su p p ly o f Na2S*9H2O, i n ­
c re a s e d i n number d u rin g th e f i r s t few d a y s , b u t l a t e r de­
c rea sed .
T h is may have b een due to th e e x h a u s tio n o f th e
20
TABLE 17
T r a n s f e r No. 3 C ounts o f S u lp h u r
O rganism s Showing E f f e c t o f Na^S• 9H20 on Growth
C u ltu r e s r e c e iv in g Na2S ^ H 2O a t 3 day i n t e r v a l s
Sample I
Sample 5
D ate
Number
z 103
Log.
Number
x 10 3
Log.
1 0/21
10/23
10/26
1 0/23
10/30
1 1 /2
1 1 /5
1 1 /8
11/10
11/12
339
1 ,0 7 3
1 ,1 9 9
3 ,3 9 0
4 ,2 3 8
3 ,3 3 3
2 ,4 8 6
2 ,2 6 0
1 ,6 2 8
707
5 .5
6 .0
6 .1
6 .5
6 .6
6 .5
6 .4
6 .3
6 .2
5 .8
621
1 ,5 8 2
1 ,8 0 8
4 ,0 6 8
4 ,9 7 2
4 ,8 5 9
3 ,8 9 8
3 ,3 9 0
3 ,2 7 7
2 ,4 8 6
5 .8
6 .2
6 .3
6 .6
6 .7
6 .7
6 .6
6 .5
6 .5
6 .4
Sample 7
Number
x 103
84
226
339
678
904
678
565
565
452
197
Log.
4 .9
5 .4
5 .5
5 .8
5 .9
5 .8
5 .8
5 .8
5 .7
5 .3
C u ltu r e s r e c e iv in g Na2S . 9H20 o n ly a t s t a r t
1 0 /2 1
1 0 /2 3
10/26
1 0/28
10/30
1 1 /2
H /5
1 1 /8
1 1/10
11/12
367
960
734
621
508
339
282
226
113
169
5 .6
5 .9
5 .9
5 .8
5 .7
5 .5
5 .5
5 .4
5 .1
5 .2
593
1 ,2 4 3
1 ,2 4 3
881,
565
395
452
452
452
452
5 .8
6 .1
6 .1
5 .9
5 .8
5 .6
5 .6
5 .6
5 .6
5 .6
113
169
226
169
113
113
56
56
—
5 .1
5 .2
5 .4
5 .2
5 .1
5 .1
4 .7
4 .7
—
21
F ig u re 3. T r a n s f e r No. 3 E f f e c t o f NagS'SHgO on grow th
o f s u lp h u r b a c t e r i a i n an in o r g a n ic medium. Red i n d i c a t e s
c u l t u r e s r e c e iv in g Na2S a t t h r e e day i n t e r v a l s . B lue
i n d i c a t e s c u l t u r e s r e c e iv in g Na2S o n ly a t th e s t a r t .
22
su p p ly o f s u lp h id e s , r e s u l t i n g i n th e o x id a tio n o f th e g r a n u le s
and e v e n tu a lly c a u s in g th e d e a th o f th e o rg a n ism .
Ten sam p les w ere c a r r i e d th ro u g h o u t th e e n t i r e i n v e s t i ­
g a tio n ; how ever, d a ta w ere k e p t o n ly on sam p les I , 5 , and 7 ,
r e p r e s e n tin g th e d i f f e r e n t pH r a n g e s , s in c e o rg an ism s fo u n d
i n a l l te n sam p les w ere s i m i l a r i n m orphology and e x h ib ite d
s i m i l a r grow th c h a r a c t e r i s t i c s .
D ata g a th e re d i n t h i s i n v e s t i g a t i o n seem to i n d i c a t e t h a t
sodium s u lp h id e i s an e s s e n t i a l m e ta b o l ite f o r the g ro w th and
c u l t i v a t i o n o f s u lp h u r b a c t e r i a .
I n th e ab se n c e o f t h i s s u l ­
p h u r compound, th e o rg a n ism s a r e n o t a b le t o c a r r y on th e
m e ta b o lic p r o c e s s e s n e c e s s a r y f o r c o n tin u e d g ro w th and r e ­
p r o d u c tio n .
A r e c e n t com m unication from D r. van K i e l ’ s
l a b o r a to r y c o n firm s t h i s and a l s o recommends th e a d d i t i o n o f
m a la te to prom ote f a s t e r and b e t t e r g ro w th .
PUB2 CULTUEH TECHNICS EMPLOYED
I t was f e l t t h a t p u re c u l t u r e s w ere n e c e s s e r y i n o r d e r
to make f u r t h e r s t u d i e s on th e s u lp h u r b a c t e r i a ; t h e r e f o r e , a
number o f p u re c u l t u r e te c h n i c s w ere t r i e d .
Van N ie l (1 9 3 0)
s t a t e s t h a t p u re c u l t u r e s can be o b ta in e d th ro u g h a s e r i e s o f
d i l u t i o n s and t r a n s f e r s .
How ever, th e a u th o r had no s u c c e s s
i n o b ta in in g p u re c u l t u r e s by t h i s m ethod.
I n none o f th e
T r a n s f e r s I , 2 , o r 3, w ere th e s e s u lp h u r o rg an ism s fo u n d in
p u re c u l t u r e .
I n T r a n s f e r No. I , th e la r g e ro d shaped s u lp h u r
o rg an ism s w ere p re d o m in a n t.
However a s s u c c e s s iv e t r a n s f e r s
23
w ere m ade, o th e r fo rm s in c r e a s e d i n number u n t i l i n T r a n s f e r
No. 3 , th e y w ere p re d o m in e n t, a lth o u g h th e r e w ere s t i l l l a r g e
numbers o f th e t y p i c a l s u lp h u r b a c t e r i a p r e s e n t .
A ll o th e r
fo rm s, a lth o u g h s t a r t i n g o u t slo w ly e v e n tu a lly overcrow ded th e
s u lp h u r o rg a h ism s.
A tte m p ts w ere made to s e c u re p u re c u l t u r e s
o f s u lp h u r b a c t e r i a from D r. van N ie l f o r f u r t h e r s t u d i e s , b u t
none w ere r e c e iv e d i n tim e t o be in c lu d e d i n th e s e s t u d i e s .
V a rio u s c h e m ic a ls have been found v a lu a b le i n g e n e r a l
b a c t e r i o l o g i c a l work f o r s e l e c t i v e l y i n h i b i t i n g o r d e s tr o y in g
one ty p e o f o rg a n ism w h ile p e r m ittin g a n o th e r ty p e to d e v e lo p .
R e p r e s e n ta tiv e c h e m ic a ls used i n th e s e i n v e s t i g a t i o n s to t r y
t o i n h i b i t o r e lim in a te o th e r fo rm s o f grow th w ith o u t a f f e c t ­
in g th e gro w th o f th e s u lp h u r b a c t e r i a w ere:
th e m e t a l l i c
compounds, m e rc u ric c h l o r id e and c u p r ic s u lp h a te ; R o d alo n , a
q u a te r n a r y ammonium compound; B-IC c h lo r in e pow der, a h y p o c h lo ra t e ; p o ta s s iu m t e l l u r i t e ; and c r y s t a l v i o l e t .
As can be se en from T a b le V, m e rc u ric c h l o r id e and p o t a s ­
sium t e l l u r i t e w ere th e o n ly compounds t h a t had any i n h i b i t o r y
e f f e c t on th e m ic r o f lo r a p r e s e n t i n th e c u l t u r e w ith o u t r e ­
d u c in g th e number o f t y p i c a l s u lp h u r o rg a n is m s .
However,
th e s e form s c o u ld n o t be c u t down to su ch an e x te n t t h a t a
p u re c u l t u r e o f a t y p i c a l s u lp h u r ty p e e x i s t e d .
P e rh a p s f u r t h e r s t u d i e s w ith a d d i t i o n a l c h e m ic a ls would
r e s u l t i n f in d in g a s p e c i f i c a g e n t t h a t would i n h i b i t o r d e s tr o y
th e v a r io u s form s found i n c o n ju n c tio n w ith th e s u lp h u r b a c t e r i a .
24
I n h i b i t o r y E f f e c t o f V a rio u s C h em icals
on H ic r o f lo r a o f H ot S p rin g s IV ithout E lim in a tio n
o f T y p ic a l S u lp h u r O rganism s
C hem ical
Sample I
Sam ple 5
Sam ple 7
M oderate
S lig h t
S lig h t
Hone
M oderate
S lig h t
S lig h t
None
M oderate
S lig h t
S lig h t
None
M o d erate
S lig h t
S lig h t
Hone
M oderate
S lig h t
S lig h t
None
M oderate
S lig h t
S lig h t
None
Hone
Hone
None
None
None
None
None
None
None
None
Hone
None
Hone
None
None
None
None
Hone
Hone
Hone
None
Hone
Hone
Hone
None
None
Hone
Hone
Hone
None
Hone
Hone
None
None
Hone
Hone
M e rc u ric c h lo r id e
1 :1 2 ,5 0 0
1 :2 5 ,0 0 0
1 :5 0 ,0 0 0
1 :1 0 0 ,0 0 0
P o ta ss iu m t e l l u r i t e
1 :1 2 5
1:250
1:500
1:1000
B-H C h lo rin e Powder
200 ppm
400 ppm
800 ppm
R odalon - A lk y l d im e th y l .
b e n z y l ammonium c h lo r id e
1 : 12,500
1 :2 5 ,0 0 0
1 :5 0 ,0 0 0
C u p ric S u lp h a te
1 :1 0 ,0 0 0
1 :2 0 ,0 0 0
1 :4 0 ,0 0 0
C r y s ta l V io le t
1 :2 5 ,0 0 0
1 : 50,000
1 :1 0 0 ,0 0 0
25
J
CLASSIFICATION OT OEGAKLSLS TOtJIID
Only a s m a ll number o f t y p i c a l s u lp h u r b a c t e r i a have been
s tu d ie d i n p u re c u l t u r e .
As a r e s u l t , th e d e s c r i p t i o n s o f
g e n e ra and s p e c ie s r e s t m a in ly on o b s e r v a tio n s made w ith c o l ­
l e c t i o n s from n a t u r a l s o u rc e s o r cru d e c u l t u r e s .
A number o f
i n v e s t i g a t o r s (W inogradsky, Cohn e t c . ) h av e i m p l i c i t l y a c c e p te d
d iffe re n c e s in c e l l s iz e as a s u f f i c ie n t j u s t i f i c a t i o n fo r
e s t a b l i s h i n g in d e p e n d e n t s p e c i e s .
However, l a t e r s t u d i e s w ith
p u re c u l t u r e s o f c e r t a i n s u lp h u r b a c t e r i a W inogradsky, van
N i e l , and o th e r s have e s t a b l i s h e d beyond a d o u b t t h a t e n v iro n ­
m e n ta l c o n d itio n s , such a s c o m p o sitio n o f th e medium and d i f ­
f e r e n c e s i n te m p e r a tu r e , may e x e r t a p ro fo u n d in f lu e n c e on th e
g e n e r a l m orphology o f th e s e o rg a n ism s; t h e r e f o r e , c a r e f u l
e v a lu a tio n o f d i s t i n g u i s h i n g f e a t u r e s i s n e c e s s a r y .
The t h r e e o rg a n ism s d e s c r ib e d i n t h i s w ork w ere c o l l e c t e d
from d i f f e r e n t l o c a l i t i e s and u n d e r v a r y in g e n v iro n m e n ta l c o n d i­
tio n s .
A ll th r e e o rg an ism s a p p e a re d t o have s i m i l a r morpho­
l o g i c a l c h a r a c t e r i s t i c s , b u t c l a s s i f i c a t i o n o f th e s e o rg an ism s
c a n n o t be d e f i n i t e b e c a u se o f th e i n a b i l i t y to o b ta in p u re
c u ltu re s .
From m eager d e s c r i p t i o n s g iv e n i n B e rg e y 1S M anual,
i t a p p e a rs t h a t th e y can be c l a s s i f i e d u n d er O rd er I ,
D u b a c t e r i a l e s . S u b o rd e r I I I , H h o d o b a c te r iin e a e . F am ily I ,
T h io rh o d ace ae (M o lis h ).
The o rg an ism s c l a s s i f i e d u n d er F am ily
T h lo rlio d aceae p ro d u ce p ig m en t sy stem s composed o f g re e n
b a c te r io c h lo r o p h y l l and y e llo w and r e d c a r o t e n o id s .
As a
26
r e s u l t , th e y a p p e a r a s p a le p u r p le , b ro w n ish to deep re d c e l l
m a sse s.
I t i s p o in te d o u t i n th e s i x t h e d i t i o n o f B e rg e y 1s
Manual t h a t pigm ent p r o d u c tio n i s d ep en d en t upon e n v iro n m e n ta l
c o n d i tio n s .
I n th e c a se o f t h e o rg an ism s s tu d ie d i n th e p r e s e n t
w ork, no p ig m e n ta tio n was n o tic e d i n c u l t u r e s grown i n a sy n ­
t h e t i c medium, a lth o u g h c o l o r s r a n g in g from l i g h t brown to
deep r e d w ere o b se rv e d i n th e sed im en t o f th e o r i g i n a l sam ples
c o lle c te d .
T h is d i f f e r e n c e i n p ig m e n ta tio n h a s b een a t ­
t r i b u t e d t o th e i n a b i l i t y o f th e i n v e s t i g a t o r to d u p lic a t e
th e e x a c t e n v iro n m e n ta l c o n d itio n s o f th e n a t u r a l h a b i t a t .
The t y p i c a l s u lp h u r o rg an ism s s tu d ie d i n t h i s i n v e s t i ­
g a t io n w ere rod shaped o rg a n ism s, o c c u rin g s i n g l y , and ra n g in g
from 4 .0 to 1 1 .0 m ic ro n s i n le n g th and from 1 .5 to 2 .0 m ic ro n s
i n w id th .
M easurem ents w ere made from c a r b o l f u c h s in s t a i n s
and a l s o sodium n i t r o p r u s s i d e s t a i n s o b se rv e d u n d er th e o i l
im m ersion o b j e c t i v e .
C e ll m orphology o f th e s e o rg a n ism s i s
shown i n P l a t e I , f i g u r e s I , 2 , and ) .
Vhen s ta in e d w ith
H u ck e r1S m o d if ic a tio n o f th e gram s t a i n , th e o u t l i n e o f th e
c e l l ap p e ared f a i n t l y gram n e g a tiv e w h ile t h e g r a n u le s w ith in
th e c e l l showed a s tr o n g gram n e g a tiv e r e a c t i o n .
Ho en d o sp o re s
w ere found a s d e te rm in e d by th e C o rn er sp o re s t a i n , n o r was
th e r e any i n d i c a t i o n o f a c a p s u le p r e s e n t when s t a i n e d by
means o f th e H is s c a p s u la r s t a i n .
No m o t i l i t y was e x h ib ite d
by th e s e o rg a n ism s when o b se rv e d u n d er th e h a n g in g d ro p p r e p ­
a ra tio n .
The p r e s e n c e o f s u lp h u r g r a n u le s w i t h i n th e c e l l
27
w a ll was d e m o n stra te d by th e u se o f th e d i f f e r e n t i a l s t a i n i n g
te c h n ic d ev elo p ed by Howard (1948) w hich em ploys sodium n i t r o p r u s s id e a s an i n d i c a t o r f o r s u lp h u r co m p lex es.
T here was no
r e l a t i o n betw een th e number o f g r a n u le s p r e s e n t w ith in th e
c e l l and th e s i z e o f th e o rg an ism , th e number v a r y in g from
two to s i x g r a n u le s .
S in c e th e o rg an ism s s tu d ie d co u ld n o t be o b ta in e d i n p u re
c u l t u r e , and s in c e a l l d e s c r i p t i o n s o f t h e s e o rg an ism s w ere
m eag er, i t was n o t p o s s i b l e t o c a r r y c l a s s i f i c a t i o n f u r t h e r
th a n O rder I , E u b a c t e r l a l e s . S u b o rd e r I I I , R h o d o b a d te r iln e a e .
F am ily I , T h io rh o d a c e a e ,
28
DISCUSSION
I t h a s been shown i n th e s e i n v e s t i g a t i o n s t h a t sodium
s u lp h id e i s an e s s e n t i a l m e ta b o l ite f o r th e c u l t i v a t i o n and
grow th of s u lp h u r o rg a n is m s .
However, th e r o l e w hich h y d ro ­
gen s u lp h id e p la y s i n th e m e ta b o lism o f s u lp h u r b a c t e r i a h a s
n o t been e s t a b l i s h e d d e f i n i t e l y .
The te rm ” s u lp h u r b a c t e r i a ” h a s b een u se d t o in c lu d e
b o th th e n o n -p ig m en ted c h e m o sy n th e tic ty p e s and th e pigm ented
p h o to s y n th e tic t y p e s . . B oth g ro u p s a re a b le t o o x id iz e h y d ro ­
gen s u lp h id e to s u lp h u r and s to r e th e s u lp h u r i n th e form o f
g r a n u le s w ith in th e c e l l s .
No m echanism o f h y d ro g en s u lp h id e u t i l i z a t i o n h a s been
p ro p o sed i n t h i s p a p e r; h o w ev er, a number o f t h e o r i e s have
been p o s t u l a t e d by o th e r w o rk e rs a s to th e p a r t p la y e d by
hydrogen s u lp h id e i n th e m e ta b o lism o f t h i s g ro u p o f o rg a n ism s.
An a tte m p t to e x p la in th e r o l e w hich h y d ro g en s u lp h id e p la y s
i n th e m e tab o lism o f c o l o r l e s s s u lp h u r b a c t e r i a was f i r s t
made by W inogradsky (1 3 8 8 ).
C u l t u r a l e x p e rim e n ts showed t h a t
th e a c c u m u la tio n o f s u lp h u r d r o p l e t s w ith in th e c e l l w a ll o f
th e s u lp h u r o rg an ism s was d e p e n d en t upon th e p r e s e n c e o f
hydrogen s u lp h id e i n th e e x t e r n a l en v iro n m e n t.
Upon d e p le tio n
o f t h i s s u lp h u r compound, th e d r o p l e t s w ith in th e c e l l would
g r a d u a lly d is a p p e a r and th e s u lp h u r - f r e e c e l l s would soon d ie
o f f u n le s s p ro v id e d w ith a f r e s h su p p ly o f s u lp h i d e .
"Wino­
g ra d sk y t h e r e f o r e p ro p o sed th e th e o ry t h a t th e s u lp h u r o rg an ism s
29
b elonged t o th e a u to tr o p h ic group o f o rg a n ism s and o b ta in e d
en e rg y n e c e s s a r y f o r grow th by o x id iz in g in o r g a n ic m a tte r , i n
t h i s i n s t a n c e h y d ro g en s u lp h id e .
Such a r e a c t i o n c o u ld be
r e p r e s e n te d by th e f o llo w in g e q u a tio n :
2EgS
4
O2
---------------- -
23 4 SH2O t en erg y
He p o s tu l a te d t h a t e n e rg y o b ta in e d from t h i s o x id a tio n c o u ld
be u t i l i z e d by th e c e l l s f o r th e r e d u c tio n o f carb o n d io x id e
to o rg a n ic p r e c u r s o r s and f o r a l l o th e r c e l l a c t i v i t i e s .
The
s u lp h u r , s to r e d i n th e form o f g lo b u le s w ith in th e c e l l w a ll,
co u ld a c t a s a r e s e r v e r e d u c in g a g e n t and be u t i l i z e d when
h y d ro g en s u lp h id e was no lo n g e r a v a i l a b l e .
T h is s u lp h u r would
th e n be o x id iz e d to y i e l d en e rg y f o r th e c e l l s a s r e p r e s e n te d
by th e e q u a tio n :
S
4
SHgO
t
302 ----------- > 2H2S0/Ji
4
6H2O
4
E nergy
The pig m en ted s u lp h u r b a c t e r i a p o se a new p ro b lem , how­
ev e r.
E v er s in c e 1883, th e p u r p le b a c t e r i a have been th o u g h t
to p o s s e s s a p h o to c h e m ic a l m e ta b o lism s im ila r to t h a t o f
g re e n p l a n t s ; how ever, i t h a s been o n ly w ith in th e p a s t te n
y e a rs t h a t any d e f i n i t e know ledge o f t h e i r m e ta b o lism and
n u t r i t i o n h a s b een o b ta in e d .
Van N ie l (1 9 4 1 ), i n com paring
th e m etab o lism o f p u r p le b a c t e r i a w ith t h a t o f g re e n p l a n t s ,
r e p r e s e n t s th e p r o c e s s o f p l a n t p h o to s y n th e s is by th e f o llo w ­
in g :
(1)
4 (H2O
4
(2)
4H
CO2
4
hv
»
H
>
(CH2O)
4
OH)
4
H2O
30
(3)
x ixdid
e --------2 (2 0 H --------- P•*»ePr oe ro
e
♦ O2
I n c o n t r a s t , he p o s t u l a t e s t h a t b a c t e r i a l p h o to s y n th e s is may
be in d ic a t e d a s fo llo w s:
(I)
—
H
111
Ocxi
0
+
(3)
'9
(2)
4 (H2O ♦ hv
2 (20H + H2A —
OH)
(CH2O) -* H2O
-------------- *
2H20 V
A)
w here hv is 3 r a d i a n t en erg y and H2A i s u s u a l l y H2S .
I n b o th
p r o c e s s e s , p h o to s y n th e s is in v o lv e s th e p h o to c h e m ic a l s p l i t t i n g
o f w a te r w ith th e a id o f some l i g h t a b s o rb in g a g e n t a s c h lo r o ­
p h y l l o r b a c te r io c h lo r o p h y l l and unknown enzyme sy ste m s. ( I ) .
A lso i n b o th , th e l i b e r a t e d h y d ro g en th e n r e d u c e s th e CO2 i n
th e atm o sp h ere t o c o n v e rs io n p r o d u c ts o f CO2 . (2)
T hus,
a c c o rd in g t o van N i e l , p l a n t and b a c t e r i a l p h o to s y n th e s e s
d i f f e r o n ly i n e q u a tio n ( 3 ) .
I n b a c t e r i a l p h o to s y n th e s is ,
th e p r o c e s s o f p h o to c h e m ic a l s p l i t t i n g may o c c u r a t lo n g e r
wave le n g th s th a n f o r p l a n t p h o to s y n th e s is r e s u l t i n g i n a l e s s ­
ened amount o f e n e rg y a v a i l a b l e to th e b a c t e r i a .
T h is e n e rg y
l e v e l may be so low t h a t i t would be im p o s s ib le f o r th e h y d ro x y la te d system to become r e a r r a n g e d i n t o a p e ro x id e compound
w ith a su b se q u e n t sp o n ta n e o u s r e g e n e r a tio n o f oxygen and w a te r
a s th e p r o c e s s o c c u rs i n p l a n t p h o to s y n t h e s is .
T h u s, i n th e
c a se o f b a c t e r i a l - p h o t o s y n t h e s i s , th e h y d ro x y l a t e d sy stem co u ld
r e g e n e r a te w a te r o n ly w ith th e a s s i s t a n c e o f a re d u c in g sy ste m .
I n t h i s c a s e , h y d ro g en s u lp h id e would f u n c tio n a s a h y d ro g en
donor ( 3 ) .
Van I I i e l r e p r e s e n t s th e e n t i r e p r o c e s s by th e
31
e q u a tio n !
CO2 + ZH2S ------------------->
(CH2O) + H2O + ZS
As d e te rm in e d from th e g e n e r a l c h a r a c t e r i s t i c s and i n f o r ­
m a tio n g a th e re d d u rin g t h i s w ork, i t a p p e a rs t h a t th e o rg an ism s
s tu d ie d b elo n g i n O rder I , E u b a c t e r i a l e s . S u b o rd e r I I I . Rhodob a c t e r l i n e a e . F am ily I , T h io rh o d a c e a e b e c a u se o f t h e i r mor­
p h o lo g ic a l and grow th c h a r a c t e r i s t i c s .
The f a m ily T h lo -
rh o d a c e a e p ro d u ce a p ig m en t sy stem composed o f g re e n b a c t e r i o c h l o r o p h y l l and y e llo w and re d c a r o t e n o id s .
As a r e s u l t ,
th e y a p p e a r a s p a le p u r p le t o deep re d c e l l m a sse s.
I n th e
p r e s e n t i n v e s t i g a t i o n s , t h i s r e d c o lo r in g was n o tic e d i n th e
se d im en t o f th e o r i g i n a l sam p les c o l l e c t e d a f t e r a two week
in c u b a tio n p e r io d .
th e t h r e e t r a n s f e r s .
H ow ever, th e c o l o r was n o t found i n any o f
T h is may h av e b een due t o i n s u f f i c i e n t
number o f o rg a n ism s p r e s e n t i n th e c u l t u r e , s in c e van N ie l
s t a t e s t h a t c o l o r i s o b se rv e d o n ly when th e o rg an ism s a r e
p r e s e n t i n la r g e c e l l m a sses and t h a t t h i s c o l o r c a n n o t be
o b se rv e d i n th e i n d i v i d u a l c e l l s .
32
SimLaRY
I n v e s t i g a t i o n s w ere c a r r i e d o u t to d e te rm in e a s a t i s ­
f a c t o r y method o f c u l t i v a t i n g s u lp h u r o rg an ism s u s in g sam p les
c o l l e c t e d from th e rm a l w a te rs o f Y ello w sto n e N a tio n a l P a rk .
1.
A c h r o n o lo g ic a l re v ie w o f th e l i t e r a t u r e on s u lp h u r
b a c t e r i a h a s been p r e s e n te d .
2.
D ata g a th e r e d i n t h i s i n v e s t i g a t i o n i n d i c a t e t h a t
sodium s u lp h id e i s an e s s e n t i a l m e ta b o lite f o r th e
grow th and c u l t i v a t i o n o f s u lp h u r b a c t e r i a .
I n th e
ab sen ce o f t h i s s u lp h u r compound, th e o rg an ism s a r e
n o t a b le to c a r r y on th e m e ta b o lic p r o c e s s e s n e c e s s a r y
f o r c o n tin u e d g ro w th and r e p r o d u c tio n .
3.
A tte m p ts w ere made to o b ta in p u re c u l t u r e s th ro u g h
a s e r i e s o f t r a n s f e r s and h ig h d i l u t i o n s , b u t w ith
no s u c c e s s .
The s u lp h u r o rg an ism s b e in g s tu d ie d
w ere g r a d u a l ly overcrow ded by o th e r fo rm s o f m ic ro ­
f l o r a p r e s e n t i n th e c u l t u r e s .
4.
A number o f r e p r e s e n t a t i v e c h e m ic a ls w hich a r e known to
have an i n h i b i t o r y e f f e c t upon c e r t a i n b a c t e r i a w ere
used i n an a tte m p t to o b ta in p u re c u l t u r e s .
None o f
th e s e c h e m ic a ls p ro v ed t o be i n h i b i t o r y to th e v a r io u s
form s found i n c o n ju n c tio n w ith th e s u lp h u r b a c t e r i a .
5.
The o rg a n ism s s tu d ie d i n t h i s i n v e s t i g a t i o n a p p e a re d
to b e lo n g i n O rd er I , S u b e c t e r l a l e s t S u b o rd e r I I I ,
R h o d o b a o te r iin e a e , F am ily I , T h io rh o d a c e a e
33
LITERATURE CITED MD CONSULTED
B a a s-B e c k e rin g , L.O.M. 1925
Ann. B o t ., 3 9 :6 1 3 .
B e rg e y , D.H.
1919
S tu d ie s on S u lp h u r B a c t e r i a .
T h e rm o p h ilic B a c te r i a .-
J . B a c t . , ZkO O l.
B ergey, D.H. 1946 M anual o f D e te rm in a tiv e B a c te r io lo g y ,
6th Edition. Y,rIlliams & Tfilkins Co., Baltimore, Md.
Brown, H.D. 1923 The C h a r a c t e r i z a t i o n o f a S u lp h u r O x id iz in g
O rganism . A b st. B a c t ., 7 :3 5 6 .
B u d e r, J . 1919 J a h r b . f . w i s s . B o t. j>8:525 a s r e p o r te d by
van N i e l , C.B , 1930 M orphology and P h y s io lo g y o f th e
P u rp le and G reen S u lp h u r B a c t e r i a . A rch . M ic r o b io l.
2 :1 .
Cohn, F . 1875 U n tersu c h u n g en u b e r B a k te r ie n , I I , B e i t r .
B io l. P f l a n z . , I , H. 3, 141 a s r e p o r te d by YZaksman, S .A .
and J o f f e , J . S . 1922 M icro o rg an ism s C oncerned i n th e
O x id a tio n o f S u lp h u r i n th e S o i l . J . B a c t . , 7 :2 3 1 .
Conn, H .J . 1940 B i o lo g ic a l S t a i n s , F o u rth Ed.
P u b l i c a t i o n s , G eneva, N.Y.
B io te c h
C ram er, I n M u lle r , C. 1870 C h e m isc h p h y sik a lisc h e B e s c h r e ibung d e r Thermen von Boden i n d e r S chw eiz a s r e p o r te d
by Waksman, S .A . and J o f f e , J . S . 1922 M icro o rg an ism s
C oncerned i n th e O x id a tio n o f S u lp h u r i n th e S o i l .
J . B a c t ., 2 :2 3 1 .
D a v is , B.M. 1897 V e g e ta tio n o f th e H ot S p rin g s o f Y ellow ­
s to n e P a rk . S c ie n c e , J$:145.
E l l i s , D avid 1932 S u lp h u r B a c t e r i a .
Company, New Y ork.
Longmann G reen and
Engelm ann, Th. YZ. 1882 B o t. Z tg . ZkO :320 f f . a s r e p o r te d
by van N ie l , C .B . 1930 M orphology and P h y s io lo g y o f
th e P u rp le and G reen S u lp h u r B a c t e r i a . A rch . M ik r o b io l.
2 :1 .
F ra n c k , J . and G a ffro n , H. 1941 P h o to s y n th e s is , F a c t s end
I n t e r p r e t a t i o n s . Adv. Enzym ., 1 :1 9 9 .
34
F r o b is h e r , M. 1946 F u n d am en tals o f B a c te r io lo g y .
S au n d ers C o ., P h i l a d e l p h i a , P a .
G aughren, E .R .L .
B a c t. R e v .,
17.B.
1947 The T h e rm o p h ilic M ic ro o rg a n ism s.
1 1 :1 8 9 .
G o r tn e r , R.A. 1944 O u tlin e s o f B io c h e m is try .
Sc, S o n s, I n c . , New Y ork.
H e n r i c i , A .T . 1948 The B io lo g y o f B a c t e r i a .
and Company, C h icag o .
Jo h n W iley
D.C. H eath
Howard, R.H. 1948 A S tu d y o f th e S u lp h u r B a c te r ia o f th e
Hot S p rin g s o f Y ello w sto n e N a tio n a l P a r k . M ontana
S t a t e C o lle g e T h e s is U n p u b lish e d ,
Im s e n e c k i, A. 1945 On th e S t r u c t u r e o f A n a e ro b ic B a c te r ia
J * B a c t . , 4 9 :1
Im s e n e c k i, A. 1945 The Growth o f A ero b ic T h e rm o p h ilic
B a c te r i a . J . B a c t . , 4 9 :5 3 9 .
K e l l , F . 1912 B e itr a g e z u r P h y s io lo g ie d e r F a r b lo s e n
Schw efe l b a k t e r i e n , B e i t r . B i o l . P f l a n z . , I I , 235 as
r e p o r te d by Waksman, S .A . and J o f f e , J . S . 1922 M icro­
o rg an ism s C oncerned i n th e O x id a tio n o f S u lp h u r i n th e
S o i l . J . B a c t. % :231.
L a n k e s te r , R. 1873 Q u a rt. J o u r n . Mio r . S c . , 12:408 as
r e p o r te d by van N i e l , C .B . 1930 M orphology and P h y s io l­
ogy o f th e P u rp le and G reen S u lp h u r B a c t e r i a . A rch .
K ik ro b i o l . 2 : 1 *
Manual o f M ethods f o r P u re C u ltu re S tu d y o f B a c t e r i a . 1946
E d ite d by The Com m ittee on B a c t e r i o l o g i c a l T ech n ic o f
th e S o c ie ty o f A m erican B a c t e r i o l o g i s t s . B io te c h
P u b l i c a t i o n s , G eneva, N.Y.
M o lisc h , H. 1907 D ie P u r p u r b a k te r ie n nach neuen U n te r suchungen a s r e p o r te d by van N i e l , C .B . 1930 M orphol­
ogy and P h y s io lo g y o f th e P u rp le and G reen S u lp h u r
B a c t e r i a . A rc h . M ik r o b io l.,
M o rriso n , L .E . and T a n n e r, F.W. 1922 S tu d ie s on Thermo­
p h ili c B a c te ria . J . B a c t., 2^343.
M o rris o n , L .E . and T a n n e r, F.W. 1924 S tu d ie s on Thermo­
p h i l i c B a c t e r i a . B o t. G a z ., 7 7 :1 7 1 .
35
N adson, G*A. 1903 On th e S u lp h u r M ic ro o rg an ism s i n th e
G u lf o f H a p s a la . B u l l. j a r d . Bot . S t . P e te r s b u r g .
1 2 :10 2 a s r e p o r te d by TJaksman, S .A . and J o f f e , J . S ,
1922 M ic ro o rg an ism s C oncerned i n th e O x id a tio n o f
S u lp h u r i n th e S o i l . J . B a c t . , 2 :2 3 1 .
P o r t e r , J .R . 1946 B a c t e r i a l C h e m istry end P h y s io lo g y .
John W iley & S o n s, I n c . , New Y ork.
S c a g l i a r i n i , G. and M o n fo rte , G. 1934 R e a c tio n betw een
Sodium N itr o p r u s s id e and A l k a l i S u l f i d e s . A t t i a c c a d .
L i n c e i , 2 0 :4 1 ; o f C.A . 2 2 :3 6 2 2 .
S c a g l i a r i n i , G. and P r a t e s i , P . 1929 The R e a c tio n Between
N itr o p r u s s id e and S u lp h id e o f Sodium . A t t i a c c a d .
L i n c e l , 8 :7 5 ; o f C.A. 2 2 :5 7 3 .
S e t c h e l l , W.A. 1903
S c ie n c e 1 7 :9 3 4 .
The U pper T em p eratu re L im its o f L i f e .
S p o n s le r , O .L. and B a th , J .D , 1942 M o le c u la r S t r u c t u r e i n
P ro to p la s m . The S t r u c t u r e o f P ro to p la s m . A Monograph
o f th e A m erican S o c ie ty o f P la n t P h y s i o l o g i s t s . Ed.
W illia m S e i f r i z , Iowa S t a t e C o lle g e P r e s s , Ames, Iow a.
S w in g le , D.B. and W a lte r , W.G. 1947 G e n e ra l B a c te r io lo g y
D. Van N o stra n d Co. I n c . New Y ork.
U m b re it, W.W.
2 :1 5 6 .
1947
P ro b lem s o f A u to tro p h y .
B a c t. Rev.
van N i e l , C.A . 1930 M orphology and P h y s io lo g y o f th e P u rp le
and G reen S u lp h u r B a c t e r i a . A rc h . M ik r o b io l. , 2 : 1 *
van N ie l , C.A. 1941 The B a c t e r i a l P h o to s y n th e s e s and T h e ir
Im p o rtan ce f o r th e G e n e ra l P roblem o f P h o to s y n th e s is .
Adv. E nzym ., 1 :2 6 3 .
van N i e l , C.A. 1944 The C u l tu r e , G e n e ra l P h y s io lo g y , Mor­
p h o lo g y , and C l a s s i f i c a t i o n o f th e N o n -su lp h u r P u rp le
and Brown B a c t e r i a . B a c t. R e v ., 8 :1
von D e in e s , 0 . 1933 D er S to f f w e c h s e l d e r S c h w e f e lb a k te r ie h .
Die N atu rw is s , 2 1 :8 7 3 . a s r e p o r te d by S ta r k e y , R .L .
1936 F o rm a tio n o f S u lp h id e by Some S u lp h u r B a c t e r i a .
J . B a c t . , 3 3 :5 4 5 .
36
Vtaksman, S .A . 1922 M icro o rg an ism s C oncerned i n th e O xida­
t i o n o f S u lp h u r i n th e S o i l - I n t r o d u c t i o n . J". B a c t .,
2 :231 .
VTaksman, S .A . 1922 M icro o rg an ism s C oncerned i n th e O xida­
t i o n o f S u lp h u r i n th e S o i l — A S o lid Medium f o r th e
I s o l a t i o n and C u l t i v a t i o n o f T h io b a c ill u s th io o x id a n s .
J . B a c t ., 2 :6 0 5 .
'
------------------i.aksman, S .A . and J o f f e , J . S . 1922 M icro o rg an ism s Concerned
i n th e O x id a tio n o f S u lp h u r i n th e S o i l . J". B a c t . ,
Warming, 3 . 1875 V id e n sk . M e d d e le ls e r n a t u r h . f o r . K jo benhavn No. 2 0-28 a s r e p o r te d by van N i e l , C .E . 1930
M orphology and P h y sio lo g y o f th e P u rp le and G reen
S u lp h u r B a c t e r i a . A rch . M ik r o b io l., 2 : 1 .
V/eed, V/.K. 1889 The V e g e ta tio n o f Hot S p r in g s .
A m erican N a t u r a l i s t . 23:39A .
The
W inogradsky, S . 1888 B e itr a g e z u r M o rphologie und P h y s io lo g ie d e r B a k te r ie n . H e ft I Z u r M orphologie und
P h y s io lo g ie d er Schw efe l b a k t e r i e n , B o t. Z tg . 4 5 :4 8 9
a s r e p o r te d by van N ie l , C .3 . 1930 M orphology and
P h y s io lo g y o f th e P u rp le and G reen S u lp h u r B a c t e r i a .
A rch. M ik ro b io l., 2 : 1 .
37
EXPLANATION OF PLATiJ I
F ig . I a . C a rb o l f u c h s in s t a i n o f t y p i c a l s u lp h u r o rg an ism s
found i n Sample No. I , showing s u lp h u r g r a n u le s
c o n ta in e d w ith in th e c e l l w a ll .
C e lls ra n g e from
5 .0 to 1 1 .0 m ic ro n s i n le n g th by 1 .5 to 2 .0 m ic ro n s
i n w id th .
F ig . I b . Sodium n i t r o p r u s s i d e s t a i n o f t y p i c a l s u lp h u r o rg a n ­
ism s found in Sample No. I .
S u lp h u r g r a n u le s w ith in
th e c e l l w a ll s t a i n b r i g h t re d by th e a c t i o n o f
sodium n i t r o p r u s s i d e w h ile th e c e l l w a ll i t s e l f
i s s ta in e d l i g h t g re e n by th e m a la c h ite g r e e n .
C e lls ran g e from 5*0 to 1 1 .5 m ic ro n s i n le n g th by
1 .5 to 2 .0 m ic ro n s i n w id th .
F ig . 2 a . C a rb o l f u c h s in s t a i n o f t y p i c a l s u lp h u r o rg an ism s
found i n Sample No. 5» show ing s u lp h u r g r a n u le s
c o n ta in e d w ith in th e c e l l w a ll .
C e lls ra n g e from
7 .0 to 1 1 .0 m ic ro n s i n le n g th by 1 .5 to 2 .0 m ic ro n s
in w id th .
F ig . 2 b . Sodium n i t r o p r u s s i d e s t a i n o f t y p i c a l s u lp h u r o rg a n ­
ism s found i n Sample No. 5 .
S u lp h u r g r a n u le s w ith in
th e c e l l w a ll s t a i n b r i g h t re d by th e a c tio n o f
sodium n i t r o p r u s s i d e w h ile th e c e l l w a ll i t s e l f
i s s ta in e d l i g h t g re e n by th e m a la c h ite g r e e n .
C e lls ra n g e from 6 .0 t o 1 4 .0 m ic ro n s i n le n g th by
1 .5 to 2.0 m ic ro n s i n w id th .
38
Fig. 3 a . Carbol fuchsin stain of typical sulphur organisms
found in Sample No. 7 , showing sulphur granules
contained within the cell wall.
Cells range from
4 .0 to 8 .0 m ic ro n s i n le n g th by 1 .5 to 2 .0 m ic ro n s
i n w id th .
F ig . 3 b . Sodium n i t r o p r u s s i d e s t a i n o f t y p i c a l s u lp h u r o rg a n ­
ism s found i n Sam ple No. 7«
S u lp h u r g r a n u le s w ith in
th e c e l l w a ll s t a i n b r i g h t re d by th e a c t i o n o f
sodium n i t r o p r u s s i d e w h ile th e c e l l w a ll i t s e l f i s
s ta in e d l i g h t g re e n by th e m a la c h ite g re e n .
C e lls
ra n g e from 5 .0 to 1 0 .0 m ic ro n s i n le n g th by 1 .5 to
2 .0 m ic ro n s i n w id th .
P la te
I
F ig u re 2
F ig u re 3
I cm - 10 m ic ro n s
MONTANA STATE OWIVCTSnV LIBRARIES
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