1964, 12(2):173. Appl. Microbiol.

Charles L. Duncan and Arthur R. Colmer

Plants and Juices

Coliforms Associated With Sugarcane
http://aem.asm.org/content/12/2/173
Updated information and services can be found at:
These include:
CONTENT ALERTS
more articles cite this article),
Receive: RSS Feeds, eTOCs, free email alerts (when new
http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:
http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:

o
n

S
e
p
t
e
m
b
e
r

1
9
,

2
0
1
4

b
y

g
u
e
s
t
h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m


o
n

S
e
p
t
e
m
b
e
r

1
9
,

2
0
1
4

b
y

g
u
e
s
t
h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

APPLIED MICROBIOLOGY
V o l . 1 2 , N o . 2 , p . 1 7 3 - 1 7 7 March, 1 9 6 4
Co p y ri g ht ( ) 1 9 6 4 Am e ri can S o ci e t y f o r Mi cro b i o l o g y
Pri n t e d i n U . S . A.
Co l i f o rm s As s o ci at e d W i t h S u g arcan e Pl an t s an d J u i ce s
CH ARLES
L.
Dt T N CAN
AN D ART H U R R. COLI\ER
De p art m s l e n t o J Bact e ri o l o g y an d Ag ri cu l t u ral Ex p e x i m e n t S t at i o n , Lo u i s i an a S t at e t U n i v e rs i t y , Bat o n l Ro t u g e , Lo u i s i at n a
Re ce i v e d f o r p u b l i cat i o n i 9 De ce m i i b e r 1 9 6 3
ABS T RACT
DU N CAN , CH ARLES L. ( Lo u i s i an a S t at e U n i v e rs i t y , Bat o n
Ro u g e ) , AN D ART H U R R. COLMER. Co l i f o rm s as s o ci at e d w i t h
s u g arcan e p l an t s an d j u i ce s . Ap p l . Mi cro b i o l . 1 2 : 1 7 3 - 1 7 7 .
1 9 6 4 . - A s t u d y o f t he co l i f o rm s f o u n d o n s u g arcan e p l an t s
t hro u g ho u t t he g ro w i n g s e as o n i n d i cat e d t hat m e m b e rs o f t he
g e n u s Ae ro b act e r co m p ri s e a p art o f t he e p i p hy t i c m i cro f l o ra.
W he n q u an t i t at i v e d e t e rm i n at i o n s w e re m ad e o f t he co l i f o rm s
b y m e an s o f t he m o s t l ) ro b ab l e n u m b e r t e chn i q u e , n o
co m p l e t e d
t e s t s w e re o b t ai n e d o n y o u n g s u g arcan e p l an t s d u ri n g t he t e s t
p ) e ri o d
o f
F e b ru ary t hro u g h MAIay ; ho w e v e r, Paraco l o b act ru m n
ae ro g e n o i d e s w as i s o l at e d f re q u e n t l y . N i n e d i f f e re n t IMV i C
t y p e s o f co l i f o rm s w e re i d e n t i f i e d am o n g t he 1 9 8 i s o l at e s cho s e n
f o r s t u d y . T he p re d o m i n at i n g co l i f o rm f o u n d at t he
t o l ) , m i d d l e ,
an d b o t t o m p o rt i o n s o f t he s u g arcan e p l an t s w as A. cl o acae , b u t
t he t y l ) e p re d o m i n at i n g i n l e af - s he at h w at e r w as A. ae ro g e n e s .
On l y o n ce w as Es che ri chi a co l i f o u n d as s o ci at e d w i t h t he p l an t s
at t he hi g h
d i l u t i o n s n e ce s s ary f o r d e t e rm i n i n g m o s t p ro b ab l e
n u m b e rs . T he n u m b e rs o f d i f f e re n t co l i f o rm s o ccu rri n g i n s u g ar-
can e j u i ce w e re g e n e ral l y d i v i d e d e q u al l y am o n g A. ae ro g e n e s ,
A. cl o acae , an d E. co l i . MIo re v ari e t y am o n g t he 1 : A\IV i C t y p e s
s e e m e d t o o ccu r o n t he can e l ) l an t s t han i n t he l e af - s he at h w at e r
o r t he can e j u i ce i n t he m i l l .
N u m n e ro u s i n v e s t i g at o rs hav e i n d i cat e d t hat t he ( o l i -
f o rm i i g ro u p o f b act e ri a, e s p e ci al l y t ho s e o f t he Ae ro b act e r
t y p e , co n s t i t u t e a p art o f t he m i cro f l o ra o f g re e n p l an t s
( Al l e n ai n d H ai ri s o t n , 1 9 : 3 6 ; T ho m as an d AMcQ u i l l i n , 1 9 5 2 ;
T ho m as an d H o b s o n , 1 9 5 5 ; K ro u l i k , Bu rk e y , an d W i s e -
i i m an , 1 9 - 5 . 5 ; F ras e r, Re i d , an d AMal co l m n , 1 9 5 6 ) . In a re -
ce n t s t u d y o n t he t y p e d i s t ri b u t i o n o f co l i f o rm b act e ri a
o n v e g e t at i o n , Ge l d re i ch, H u f f , an d Be s t ( 1 9 6 3 ) f o u n d a
t o t al o f 1 3 L\IV i C t y p e s o n 3 0 s p e ci e s o f p l an t s , w i t h n o n e
o f t he IM1 V i C t y p e s ap p e ari n g t o p re d o m i n at e i n an y o n e
are a cho s e n f o r s am p l i n g .
T he p re s e n ce o f an i n d i g e n o u s , e p i p hy t i c m i cro f l o ra o n
s u g arcan i e p l an t s w as as ce rt ai n e d b y W o l z o g e n K i u hr' s
( 1 9 2 3 ) an d M\Iay e u x ' s ( 1 9 6 0 ) e x p e ri m e n t s . Ge e rl i g s ( 1 9 2 4 )
co n f i rm l i e d t hat t he can e arri v i n g at t he s u g arm i l l co n t ai n e d
an e x t e n s i v e m i cro f l o ra o n i t s s u rf ace , b u t he g av e n o
d e t ai l s as t o t he n at u re o f t he o rg an i s m s . H u t chi s o n an d
Ram ay y ar ( 1 9 2 5 ) i s o l at e d f ro n m f re s h can e p l an t s As p e r-
g i l l u s s p e ci e s an d y e as t s o f t he S accharo m n y ce s ce re v i s i ae
t y p e , b u t i t w as s u s p e ct e d t hat t he o ri g i n w as e i t he r f ro i m
t he s o i l ad he ri n g t o t he can e o r f ro i m w o u n d s o f t he can e
s t al k i n f l i ct e d b y can e b o re rs .
T he n u m b e rs o f v i ab l e b act e ri a o ccu rri n g i n f re s hl y
e x t ract e d can e j u i ce s w e re d e t e rm - i n i e d b y Ow e n ( 1 9 1 1 ) ,
K o p e l o f f an d K o p e l o f f ( 1 9 1 9 ) , l e d e rs o n ( 1 9 3 8 ) , an d
i \Ii l l s t e i n , T o b i n , an d _ McCl e s k e y ( 1 9 4 1 ) , t he l at t e rw o rk e rs
p ay i n g p art i cu l ar at t e n t i o i n t o t he p re s e n ce o f Le u t co n o s t o c
i ) i e s e n t e ro i d e s .
Al t ho u g h _ May e u x ' s ( 1 9 6 0 ) e x p e ri m i e n t s s ho w e d t he
p re s e n ce o f co l i f o rm s o n can e p l an t s , i n t he s o i l ab o u t t he
s t o o l , an d i n t he j u i ce i n p o rt i o n s o f t he m i l l , n o at t e m i i p t
w as i m i ad e t o re l at e t he co l i f o rm s t o t he g ro w i n i g cr' o p
t hro u g ho u t a g ro w i n g s e as o n e x t e n d i n g f ro m i March
t hro u g h N o v e m b e r.
T he p re s e n t i n v e s t i g at i o n w as m n ad e p ri m i ari l y f o r t he
e n u m i i e rat i o n an d d e t e rm i n at i o n o f t he t y p e s o f co l i f o ri n
b act e ri a f o u n d as s o ci at e d w i t h t he g ro w i n g s u g arcan e
p l an t t hro u g h s u ch a p e ri o d an d w i t h t he j u i ce f ro m t he s e
p l an t s w he n e x p re s s e d f ro m i t he f i rs t cru s he r o f t he s u g ar
m i l l . F o r p u rp o s e s o f co m l p ari s o n , t he t o t al n u m b e rs o f
m i cro o rg an i s m i s w e re al s o d e t e rm i n e d .
. MAT ERIALS AN D CMET H ODS
S am n p l i n g . F o r e arl y s p ri n g s am p l i n g , s t e ri l e f o rce p s an i d
s ci s s o rs w e i e u s e d t o cl i p e n t i re y o u n g p l an t s ( 8 t o 1 1 0
cm hi g h) e i t he r f ro m w t he p l an t can e o f t he p re ce d i n g f al l
o r f ro m i t he n e w g ro w t h o f s t u b b l e can e . De p e n d i n g o n t he
ag e o f t he p l an t , t he cu t w as m ad e e i t he r at t he b as e o f t he
p l an t o r at v ari o u s s e ct i o n s o f i t d e p e n d i n g o n t he p l an t ' s
he i g ht . Ol d e r p l an t s ( 1 2 0 t o 2 7 0 cm 1 1 hi g h) w e re s am e p l e d b y
s e v e ri n g t he e n t i re p l an t at i t s b as e w i t h a s t e ri l e k n i f e .
S t e ri l e s ci s s o rs w e re u s e d t o cl i p t he l e av e s ab o u t 1 i n .
f ro n m t he s t al k . Lo n g s t e ri l e p ap e r b ag s i m i ad e b y f o l d i n g
an d t ap i n g b ro w n w rap p i n g p ap e r i n t o t he d e s i re d p ro -
p o rt i o n s w e re u s e d f o r t ran s p o rt i n g t he p l an t s . In t he
l ab o rat o ry , t he p l an t s w e re arb i t rari l y d i v i d e d i n t o t hre e
p o rt i o n s co m i i p ri s i n g t he t o p , m i d d l e , an d b o t t o m . T he s e
t hre e p o rt i o n s co n s t i t u t e d t hre e s e p arat e s am p l e s o f o n e
can e p l an t .
Le af - s he at h w at e r i s at i m i o s p he ri c w at e r w hi ch co l l e ct s
o n t he l e av e s o f s u g arcan e p l an t s an d i s d i re ct e d , t o g e t he r
w i t h an y p l an t e x u d at e s , i n t o t he l e af s he at h w he re i t
cl as p s t he s t al k . T he am i i o u n t o f w at e r cau g ht w i t hi n t he
s he at h i s hi g hl y d e p e n d e n t o n t he am - i o u n t o f rai n f al l ,
hu m i d i t y , an d t e m i i p e rat u re . S t e ri l e p i p e t t e s w i t h f i n e l y
d raw n e n d s w e re u s e d t o re m o v e t he w at e r, t he s he at h
b e i n g g e n t l y p u l l e d b ack t o f aci l i t at e t hi s p ro ce d u re . A
s am p l e co n s i s t e d o f w at e r f ro m 5 t o 1 6 l e af s he at hs .
S am i i p l e s o f can e j u i ce w v e re o b t ai n e d f ro m i t he f i rs t ro l l e r
1 7 3

o
n

S
e
p
t
e
m
b
e
r

1
9
,

2
0
1
4

b
y

g
u
e
s
t
h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

1 I7 U - N CAN AN - D C- ) IJ A\EA
o f t he e x p e ri m e n i t al Au d u b o n i s u g ai r m i l l o n t he Lo u i s i an a
S t at e U n i v e rs i t y cam i i p u s . T he s e s ai m i p l e s w e re co l l e ct e d i n
s t e i ri l e 6 - o z p re s cri p t i o n i b o t t l e s .
S u s p e n i s i o n s w e re m i i ad e f ro i m i y o u n g can e p l an t s an d f ro m i i
t he t o p , m i d d l e , an d b o t t o m i p o rt i o l s o f o l d e r can e p l an t s
b y b l e n d i n g 2 0 g o f p l an i t m i i at e ri al an d 1 8 0 m l o f s t e ri l e
d i s t i l l e d w at e r f o r 2 m i n i i n a W ari n g Bl e n d o r. F u rt he r
d i l u t i o n s w e re m i i ad e f ro m i i t hi s s u s p e n s i o n i . T he i n i i t i al
d i l u t i o n s o f l e af - s he at h w at e i an d can e j u i ce w e re p re p are d
b y ad d i n g
1 m l o f t he t e s t m i i at e ri al t o a 9 - m i i l w at e r b l an k .
Is o l at i o n an d d i f f e re n t i al t e s t s . A m o d i f i cat i o n o f t he MY
ag ar o f H ay n e s , W i ck e rham , ai n d H e s s e l t i n e ( 1 9 5 5 ) w v as
u s e d f o r t he p o u re d p l at e m i i e t ho d o f d e t e rm i i n i n i g t he t o t al
b act e ri al p o p u l at i o n . T he co m p o s i t i o n o f t he i m i o d i f i e d
m i i e d i u m i i w as : m l al t e x t ract , 3 g ; y e as t e x t ract , 3 g ; p e p -
t o n l e 7 ,
g ; g l u co s e , 1 0 g ; ( N H 4 ) 2 S 0 4 , 2 g ; Mg S 0 4 7 1 2 0 ,
2 g ; K 2 H PO4 , 2 g ; ag ar, 2 0 g ; an d d i s t i l l e d w at e r, 1 , 0 0 0 m l .
T he s t an d ard l act o s e b ro t h, Bri l l i an t Gre e n i l act o s e Bi l e
2 W b ro t h, Eo s i n Al e t hy l e n e Bl u e Ag ar ( EM\IB) an d
n i u t ri e n t ag ar n o rm i i al l y e m i i p l o y e d i n t he i s o l at i o n o f ai n d
t he d e t e rm i i i n at i o n o f t he i m i o s t p ro b ab l e n u m u b e r ( l \ 1 ' N )
o f co l i f o rm i s w e e re u s e d ( Am e ri can Pu b l i c H e al t h As s o -
ci at i o i , 1 9 5 5 ) . T he t e m i ap e rat u re o f i n cu b at i o n f o r t he s e
t e s t s w as 3 5 ) C.
EC m e d i u m , i n cu b at e d at t he e l e v at e d t e i i m p e rat u re o f
4 . 5 . 5 - ) C ( F i s hb e i n , 1 9 6 2 ) , w as u s e d f o r t he MIP' N d e t e rm i i i -
n at i o i n o f Es che ri chi a co l i . T u b e s w v e re re ad f o r t he p re s e n i ce
o f g as at t he e n d o f 2 4 an d 4 8 hr. An y t u b e f o u n i d p o s i t i v e
w as s t re ak e d o i n t o EAIB Ag ar an d t he n i i n cu b at e d at 8 5 C
f o i 2 4 hr. Co l o n i e s w e re p i ck e d f ro m t he s e p l at e s o n t o
n u t i ri e n t ag ar s l an t s f o r i d e i n t i f i cat i o n p u rp o s e s . T ho s e
t u b e s n o t s ho w i n g g as p ro d u ct i o n at t he e n d o f 4 8 hr at
4 5 . 5 C w e re g i v e n i ai n ad d i t i o n al 2 4 hr o f i n cu b at i o n at
3 5 C.
Ot he r m e t ho d s f o r t he d e t e rm i i i n at i o n o f i n u m i i b e rs o f
co l i f o ri m s w e re t he i i i e m i i b ran ae - f i l t e r t e chn i q u e u t i l i z i n g
n i - En d o b ro t h an d t he p o u re d p l at e m i i e t ho d u s i n g V i o l e t
Re d Bi l e ag ar.
T ' he s u b s t rat e s u s e d i n i d e n t i f i cat i o n o f t he co l i f o rm s
T ABLE 1 . YT o t al n u m i t t b e rs o f
mi cro o r- g an i s n m s an d MPN o f co l i f o rm l s
i n t he p re s u m i p t i v e an d co n f i rm t i e d t e s t s o n y o u n g
s u g
q arcan e p l an t s *
Pl an t T o t al m i cro - Pre s u m p t i v e t Co n f i rm e d t
Dat e
he i g ht
o rg an i s
) s t
( p e r 1 0 0 m l ) ( p e r 1 0 0 m l )
cm
2 / 2 3 / 6 2 8 - 1 4 2 3 3 2 2 0 . 0 2 2 0 . 0
3 / 8 / 6 2 1 5 - 2 0 1 , 8 5 0 3 3 . 0 1 7 . 0
3 / 1 6 / 6 2 2 0 - 2 5 7 , 2 0 0 4 , 9 0 0 . 0 2 2 0 . 0
3 / 2 6 / 6 2 2 5 - 3 0 7 , 5 0 0 3 . 3 0 . 0
4 / 2 / 6 2 3 5 - 4 0 2 , 2 0 0 3 5 0 . 0 0 . 5
4 / 9 / 6 2 3 5 - 5 0 2 0 7 3 . 4 0 . 8
4 / 2 7 / 6 2 6 0 - 1 0 ( ) 1 , 0 3 0 1 . 7 0 . 0
5 / 9 / 6 2 7 0 - 1 0 0 3 , 1 0 0 0 . 0 0 . 0
5 / 2 4 / 6 2 1 0 0 - 1 1 0 2 5 , 5 0 0 4 . 9 0 . 0
*
N o co m p l e t e d t e s t s w e re o b t ai n e d .
t V al u e s X 1 0 3 .
w e re D) i f co 1 \t R1 7 1 ' i m e d i u m n , K o s e r ci t l at e b ro t h, S i m m i i i i o u i s
( i t rat e ag ar, T ry p t o n e b ro t h, n u t r i e n t g e l at i i n , g l y ce ro l
b ro t h, an d t he s o d i u i i hi p p u rat e b ro t h o f H aj n i a an d
I) am o n ( 1 9 8 4 ) .
RES U LT S AN D DI) S CU S S ION
T he re s u l t s o b t ai i n e d i n t hi s s t u d y , u n l i k e t ho s e o f p i e -
v i o u s w o rk e rs , re p re s e n t an an al y s i s o f t he co l i f o ri n i f l o ra
o f acho s e n p l ai l t o f m i i ark e d ag ri cu l t u ral s i g n i f i can ce s ho rt l y
af t e r i t e m i e rg e s f ro i m i t he s o i l , t hi o u g h i t s g ro w v i n i g s e as o n i ,
an d u p t o an d i n cl u d i n g t he t i i m i e i t i s cu t ai n d harv e s t e d f o r
i n i d u s t ri al p u rp o s e s . In ad d i t i o n , n u l u i b e rs o f o t he r o r-
g an i s m i u s are g i v e e n t o i l l u s t rat e t he o ccu rre n ce o f t he co l i -
f o rm l g r' o u p re l at i v e t o t he t o t al m i cro b i al p o p u l at i o n .
Yo u n g s u g arcan e p l an t s . T he M\IP' N o f co l i f o rm i i s an d t he
t o t al n u m i i b e r o f i m i i cro o rg an i s m i i s o In y o u n g s u g ar' can i e
p l an t s are g i v e n i n T ab l e 1 . T he s e v al u e s re p re s e n t an
an al y s i s o f t he e n t i re p l an t as a s am i i p l e . It i s i n t e re s t i n g t o
n o t e t hat , f ro i i F e b ru ary t hro u g h May , n o ( o m p l e t e d
t e s t s f o r co l i f o ri m i s e - e re o b t ai n e d . T he p re s u m p t i v e an d
co n f i rl m e d M N ' N v al u e s w - e re q u i t e hi g h u i n t i l 9 Ap ri l .
It m l ay b e t hat t he s e hi g h v al u e s w hi ch w e re n o t ( o l p l e t e d
are a re f l e ct i o n o f t he s o i l - b o rn e o rg an i s m s s p l as he d u p o In
t he l o w p l an t s d u ri n g p e ri o d s o f rai n o r d u ri n g cu l t i v at i o n
o f t he p l an t s . T hi s w - o u l d e x p l ai n t he l o w e r v al u e s o b t ai t n e d
i n t he p re s u m p t i v e t e s t w he i n t he p l an t s re ache d a he i g ht
o f 5 ) 0 t o 1 0 0 cm l l , ari d at w hi ch t i m i i e t he i n f l u e n ce o f t he
s o i l N as m l u ch l e s s . Ad d i t i o n al t e s t s co n d u ct e d o n y o u n g
can e p l an t s d u ri n g a s e co i n d g ro w i n g s e as o n i g av e re s u l t s
v e ry s i m n i l ar t o t ho s e s ho w n he re .
In at t e i i i p t i n g t o d e t e rm i i i n e t he o rg an i s m i i o r o rg ai n i s i m i s
re s p o n s i b l e f o r t he p o s i t i v e p re s u m i i p t i v e an d co n f i rm e d
t e s t s w hi ch d i d n o t g i v e a co m p l e t e d t e s t , cl o s t ri d i a, as
e v i d e n ce d b y s t o rl n y f e rl u e i n t at i o n i n i ro n - f o rt i f i e d l i t m l u s
m i i i l k , w e re f o u n d t o b e p re s e n t i n al l b u t o n e o f t he ad d i -
t i o n al s am p l e s e x am i n i n e d . S i n ce e f f o rt s t o i s o l at e Baci l l u s
p o l y u n y . ra e re u n s u cce s s f u l , i t i s s u g g e s t e d t hat cl o s t ri d i a,
p o s s i b l y i n ( o n i u n ct i o n w i t h t he i n o rm i i al l y o ccu rri n g s y n e r-
g i s t i c o rg an i s m i i s f o u n d i n s u ch a v ari e d m i cro f l o ra, m i g ht
b e re s p o n s i b l e f o r t he f al s e p o s i t i v e re s u l t s .
S l o w l act o s e - f e rm i i e n t i n g o rg an i s m s i d e n t i f i e d as Para-
T ABLE 2 . Co m p ari s o n o f Ml PAN o f co l i f o rm s f o u n d at t he t o p , m l l i d d 1 l e ,
an d b o t t o m p o rt i o n s o f s u i g at can e p l an t s
Dat e Pl an t he i g ht
T o p * Mi d d l e * Bo t t o m *
( p e r 1 0 0 g ) ( p e r 1 0 0 g ) ( p e r 1 0 0 g )
cm
6 / 1 3 / 6 2 1 2 0 - 1 5 0 0 . 2 0 . 0 0 . 0
6 / 1 9 / 6 2 1 2 5 - 1 5 0 2 2 0 . 0 1 . 1 1 . 3
6 / 2 6 / 6 2 1 5 0 - 1 9 0 2 . 3 2 . 2 0 . 8
7 / 1 1 / 6 2 2 0 0 - 2 5 0 3 5 0 . 0 1 . 7 1 . 4
7 / 2 1 / 6 2 2 3 0 - 2 5 0 2 , 4 0 0 . 0 1 , 3 0 0 . 0 7 9 0 . 0
7 / 2 8 / 6 2 2 3 0 - 2 5 0 2 2 0 . 0 5 , 4 0 0 . 0 7 9 , 0 0 0 . 0
8 / 3 0 / 6 2 2 6 0 - 2 7 0 0 . 0 0 . 7 1 7 0 . 0
9 / 2 0 / 6 2 2 6 0 - 2 7 0 0 . 0 1 1 , 4 0 0 . 0 4 9 0 . 0
1 0 / 4 / 6 2 2 6 0 - 2 7 0 0 . 0 3 , 5 0 0 . 0 3 . 3
*
V t al u e s X 1 0 3 .
I 7
APPL. AIICROBIOL.

o
n

S
e
p
t
e
m
b
e
r

1
9
,

2
0
1
4

b
y

g
u
e
s
t
h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

COLIF OR- MS AS S OCIAT ED W IT H S U GARCAN E
co l o b act ru i n ae ro g e n o i d e s w e re i s o l at e d f re q u e n t l y f ro l l m t he
EA\IB p l at e s o f t he co l n f i rm n e d t e s t . T he s e o rg an i s m i i s g av e
re act i o n s i n t he v ari o u s m n e d i a u s e d f o r t he i d e n t i f i cat i o n
o f co l i f o rm s e x act l y as d i d t ho s e f o r A. ae ro g e n e s e x ce p t
f o r t he co n s i s t e n t l y d e l ay e d f e rm i e n t at i o n o f l act o s e o r,
i n s o m i i e i n s t an ce s , n o f e rm e n t at i o n at al l .
N - o co rre l at i o n i s e v i d e n t b e t w e e i n t he APN o f co l i f o rm i i s
an d t he t o t al n u m b e rs o f m u i cro o rg an i s m s . Du ri n g t he
e arl y p art o f t he g ro w i n g s e as o n , al m l - o s t al l o f t he b act e ri al
co l o i i i e s w e re y e l l o w p i g m i l e n t e d , w he re as l at e r i n t he s e as o n
t he p i g m i i e n t at i o n o f t he co l o n i e s w as m u ch m o re v ari e d .
T he t y p i cal l y o ccu rri n g y e l l o w - b act e ri u m i w as t e n t at i v e l y
i d e n t i f i e d as a F l av o b act e ri u n t .
Ol d e r s u g arcan e p l an t s . T he f i rs t t i m l e d u ri n g t he g ro w i n g
s e as o n i t hat a co m p l e t e d W PN w as o b t ai n e d w as 1 3 J u n e
( rT ab l e 2 ) . T he t o p p o rt i o n o f t he p l an t s s e e m e d t o hav e
t he g re at e s t AIPON v al u e s d u ri n g t he e arl y p art an d t he
m u i d d l e o f t he s u n m m i i e r, w he re as t he m n i d d l e an d b o t t o m n
p o r' t i o In o f t he p l an t s had hi g he r AW 1 N v al u e s t o w ard t he
l at t e r p art o f t he s u m m l l i e r an i d t he e arl y f al l . T hi s co u l d
b e a i re f l e ct i o n o f t he w e at he r co n d i t i o n s , o r t he av ai l ab i l i t y
o f i n u t t i e n i t m i at e ri al s , m n o re n u t ri e n t s b e i n g s e cre t e d at t he
n l i d d l e an d b o t t o m o f t he p l an t as t he p l an t b e cam i e o l d e r.
T he re w v as l e s s f l u ct u at i o n i n t he v al u e s f o r t o t al n u m i i b e r s o f
m i i cro o rg an i s m s ( T ab l e 3 ) t han i n t he MPN v al u e s f o r a
p art i cu l ar are a.
Le al - s he at h w at e r. May e u x ( 1 9 6 0 ) d e m i i o n s t rat e d t he
p re s e n ce o f carb o hy d rat e s ( cal cu l at e d as re d u ci n g s u b -
s t an ce s ) i n l e af - s he at h w at e r ran g i n g f ro m 0 . 0 8 t o 3 0 . 0
m g / l n l o f s am l p l e . It w o u l d s e e i i i t hat t hi s w at e r, he l d as
i t i s ag ai n s t t he n o d e o f t he s u g arcan e p l an t , w o u l d b e an
e n v i ro n m i i e n t s u i t ab l e f o r t he g ro w t h o f m i cro o rg ai n i s m i i s .
In t he p re s e n t s t u d i e s , t he A f IP' Xv al u e s ran g e d f ro l n i 0 t o
1 . 3 X 1 0 9 w i t h a g re at d e al o f v ari at i o n am i i o n g t he s am i i p l e s
( T ab l e 4 ) . S am p l e s 2 an d 8 w e re e x t re l n e l y v i s ci d an d
s l i m l l y w v - he n o b t ai n e d , i n i d i cat i n g a hi g he r n u l n b e r o f
s l i m i i e - p ro d u ci n g o rg an i s m i i s . T he AW IIN v al u e s , as w e l l as
t ho s e f o r t o t al n u m b e rs , w e re m i i u ch hi g he r i n t he s e s am n -
p l e s .
S u g arcan e j u i ce f i ro m l t he J i rs t ro l l e r o f t he m i l l . T he M\f I' N
o f co l i f o rm s i n s u g arcan e j u i ce , as d e t e rm i n e d b y s t an d ard
T ' A B I. E 3 . Co m p ari s o n o f t o t al n m u m b e rs o f m i i i cro o rg an i s m i t s f o l u n d
( i t t he t o p , m i d d l e , an d b o t t o m l p o rt i o n s o f s u i g arcan e p l an t s
Dat e Pl an t he i g ht
T o p *
Mi d d l e * Bo t t o m *
( p e r g ) ( p e r g ) ( p e r g )
cm
6 / 1 3 / 6 2 1 2 0 - 1 5 0 1 3 . 8 5 . 4 4 . 2
6 / 1 9 / 6 2 1 2 5 - 1 5 0 1 0 . 7 5 . 8 0 . 8
6 / 2 6 / 6 2 1 5 0 - 1 9 0 1 7 . 0 1 3 . 1 9 . 5
7 / 1 1 / 6 2 2 0 0 - 2 5 0 6 . 9 4 3 . 0 5 5 . 0
7 / 2 1 / 6 2 2 3 0 - 2 5 0 8 . 7 4 . 9 5 4 . 0
7 / 2 8 / 6 2 2 3 0 - 2 5 0 3 4 . 0 2 1 . 4 1 2 7 . 0
8 / 3 0 / 6 2 2 6 0 - 2 7 0 6 5 . 0 3 5 0 . 0 1 7 0 . 0
9 / 2 0 / 6 2 2 6 0 - 2 7 0 2 0 . 0 1 9 0 . 0 9 0 . 0
1 0 / 4 / 6 2 2 6 0 - 2 7 0 7 . 0 3 7 . 0 6 8 . 0
* V al u e s X 1 0 6 .
m i e t ho d s an d b y t he u s e o f EC m i i e d i u m at t he e l e v at e d
t e m i i p e rat u re o f 4 5 . 5 C, i s s ho w n i n T ab l e 5 . T he MPN
o b t ai n i e d i n t he s t an d ard t e s t , ran g i n - g f ro m a 3 5 , 0 0 0 , 0 0 0
t o 3 5 0 , 0 0 0 , 0 0 0 p e r 1 0 0 m l , w e re i m u ch g re at e r t han t he
n i u m l b e r s o b t ai n e d i n EC m e d i u m , w hi ch ran g e d f ro m
2 , 3 0 0 t o 3 5 0 , 0 0 0 p e r 1 0 0 m i l . T hi s i n d i cat e s t hat t he n u m b e r
o f Ae ) ro b act e r t y p e s are m u ch hi g he r t han t he n u n m b e r o f
Es che ri chi a t y p e s i n t he e x p re s s e d s u g arcan e j u i ce . W i t h
an l ad d i t i o n i al i i n cu b at i o n o f 2 4 hr at 3 5 C f o r t he EC
n m e d i u m i , i t can b e s e e n i t hat t he 1 \IPN v al u e s i n i e re as e d
o n l y s l i g ht l y , o r n o t at al l , as i s t he cas e i n s am p l e s 6 an d
8 . S i n i ce t he s e f i n i al v al u e s are s t i l l m i i u ch l o w e r t han t ho s e
o b t ai n i e d b y s t an d ard m e t ho d s , i t i s p o s s i b l e t hat e i t he r
t he o rg an i s m s f ai l e d t o s u rv i v e at 4 5 . 5 C o r t hat t he
e n z y m i i at i c g as - p ro d u ci n g ; m e chan i s m l s w e re i rre v e rs i b l y
i n hi b i t e d .
T he hi g he r t o t al n u m i b e rs o f m i cro o rg ai n i s m i i s i n s am i i p l e s
1 , 2 , 3 , an d 8 ( T ab l e 5 ) s e e m i i t o b e re l at e d t o hi g he r MIPN
v al u e s i n t he s e s am e s am i i p l e s . T he av e rag e p H o f t he can e
j u i ce s al u p l e s w as 5 . 4 5 . De e p - re d p i g l n i e n t e d co l o n i e s
i d e n i t i f i e d as S e rrat i a o ccu rre d f re q u e n t l y at t he hi g h
d i l u t i o n s n e ce s s ary t o o b t ai n t o t al p l at e co u i n t s . S i n ce t hi s
T ABLJ E 4 . M1 1 PN o f co l i f o rm 1 s an d t o t al n u m b e rs o f m i cro o rg an i s m s
i n l e af - s he at h w at e r
T o t al m i cro - MPN t
S am p ) l e p H o rg an i s m s * ( p e r g ) ( p e r
1 0 0
m l )
1 6 . 2 9 . 4 4 . 9
2 6 . 8 1 , 6 9 0 . 0 7 , 9 0 0 . 0
3 6 . 4 2 8 5 . 0 0 . 0
4 6 . 9 8 7 . 0 1 7 . 0
5 6 . 6 3 4 7 . 0 3 . 3
6 7 . 4 3 3 . 0 1 3 . 0
7 . 3 3 4 . 0 0 . 8
8 6 . 5 1 , 0 2 0 . 0 1 3 0 , 0 0 0 . 0
9 6 . 6 5 4 0 . 0 0 . 2
1 0 6 . 8 6 5 0 . 0 7 . 9
1 1 6 . 4 5 6 0 . 0 0 . 0
*
Aal u e s X 1 0 5 .
t
V ' al u e s X 1 0 4 .
T ABLE 5 . T o t al n u m b e rs o f m i i i cro o rg an i s m t i s an d MPN o f co l i f o rn m s
i n s u t g arcan e j u i ce as d e t e rm i n e d b y s t an d ard m e t ho d s an d b y
u s e o f EC m e d i u m at an e l e v at e d t e m p e rat u re
T o t al S t an d ard EC m e d i u m w i t h
m i cro - m u l t i p l e
EC m e d i u m
ad d i t i o n al 2 4 hr
S am p l e
o rg an i s m s *
t u b e t e s t * 4 5 . 5 C ( 4 8 hr) t o f i n cu b at i o n at
( p e r g ) ( p e r 1 0 0 m l )
( p e r
1 0 0
m l )
3 5 Ct ( p e r 1 0 0 m l )
1 5 5 3 5 0 . 0 3 5 . 0 5 4 . 0
2 3 3 7 9 . 0 2 2 . 0 1 6 0 . 0
3 6 7 3 5 . 0 1 6 0 . 0 9 2 0 . 0
4 9 3 . 5 4 . 9 7 . 9
5 1 2
-
5 . 4 3 5 . 0 5 4 . 0
6 3 9 4 . 9 2 4 0 . 0 2 4 0 . 0
7 2 2 5 . 4 4 . 9 9 2 . 0
8 3 0 5 4 . 0 3 5 0 . 0 3 5 0 . 0
9 9 1 6 . 0 4 . 9 2 4 . 0
1 0 1 8 9 . 2 2 . 3 1 3 . 0
*
V al u e s
X
1 0 6 .
t AV al u e s X
1 0 3 .
l ' OL.
1 2 Y
1 9 6 4 1 7 5

o
n

S
e
p
t
e
m
b
e
r

1
9
,

2
0
1
4

b
y

g
u
e
s
t
h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

DU N CAN AN D COLMIER
o rg an i s m w as n o t f o u n d i n an y p l at i n g s o f s am p l e s o f
s u g arcan e p l an t s , i t m ay b e t hat i t w as p i ck e d u p f ro m t he
s o i l i n t he harv e s t i n g o f t he p l an t s .
In an o t he r p o rt i o n o f t he s t u d y ( T ab l e 6 ) , a co m p ari s o n
o f t he s t an d ard m u l t i p l e t u b e t e s t , m - En d o b ro t h w i t h t he
Mi l l i p o re f i l t e r, an d V i o l e t Re d Bi l e ag ar m e t ho d s f o r
d e t e rm i n i n g n u m b e rs o f co l i f o rm s i n s u g arcan e j u i ce s ho w s
t hat t he s t an d ard t e s t g av e hi g he r MPN v al u e s t han d i d
t he o t he r t w o m e t ho d s , w i t h t he m - En d o b ro t h b e i n g
g e n e ral l y m o re s e l e ct i v e t han t he V i o l e t Re d Bi l e ag ar.
Al l co l i f o rm s i s o l at e d f ro m t he Mi l l i p o re f i l t e r w e re o f t he
Ae ro b act e r t y p e . T he f i n d i n g o f n o Es che ri chi a- t y p e o r-
g an i s m s b y t he i Ii l l i p o re f i l t e r t e chn i q u e can b e e x p l ai n e d
o n t he b as i s o f t he hi g h d i l u t i o n s n e ce s s ary t o o b t ai n
i s o l at e d co l o n i e s o n t he f i l t e r d i s cs .
An al y s i s o f d i s t ri b u t i o n o f co l i f o ri n i s o l at e s . A co m p i l at i o n
o f t he n u m b e rs o f d i f f e re n t co l i f o rm s i s o l at e d f ro m t he
v ari o u s s am p l i n g are as ( T ab l e 7 ) s ho w s t he p re d o m i n at i n g
co l i f o rm at t he t o p , m i d d l e , an d b o t t o m p o rt i o n o f t he
p l an t t o b e A. cl o acae ; y e t , t he co l i f o rn m p re d o m i n at i n g i n
l e af - s he at h w at e r w as A. ae ro g e n e s . On l y o n ce w as E. co l i
f o u n d i n co n n e ct i o n w i t h t he p l an t at t he hi g h d i l u t i o n s
n e ce s s ary f o r d e t e rm i n i n g MPN v al u e s b y s t an d ard
m e t ho d s , i n d i cat i n g t hat , al t ho u g hE. co l i m i g ht b e p re s e n t ,
i t w as n o t p re s e n t i n v e ry hi g h n u m b e rs . It i s al s o p o s s i b l e
T ABLE 6 . Co m p ari s o n o f m i e t ho d s f o r d e t e rm i n i n g n u m b e rs
o f co l i f o rm s i n s u g arcan e j u i ce
S t an d ard m u l t i p l e m - En d o b ro t h
V i o l e t Re d Bi l e
S am p l e t u b e t e s t * Mi l l i p o re f i l t e rt
ag art Re r m l )
( p e r 1 0 0 m l ) ( p e r m l ) ag art ( p e r m l )
1 3 5 0 . 0 6 2 . 0 7 1 . 0
2 7 9 . 0 4 2 . 0 3 9 . 0
3 3 5 . 0 9 . 0 9 . 0
4 3 . 5 1 . 0 0 . 8
5 5 . 4 0 . 7 0 . 6
6 5 . 4 9 . 0 1 3 . 0
7 4 . 9 2 2 . 0 7 9 . 0
8 5 4 . 0 2 3 . 0 4 3 . 0
9 1 6 . 0 2 . 5 3 . 0
1 0 9 . 2 6 . 3 5 . 9
*
V al u e s X 1 0 8 .
t V al u e s X 1 0 4 .
T ABLE 7 . Di s t ri b u t i o n o f d i f f e re n t co l i f o rn m b act e ri a
f ro m v ari o u s s am p l i n g are as *
S am p l i n g are a
Org an i s m Pl an t Pl an t Pl an t Le af - Can e
t o p m i d d l e b o t t o m s he at h j u i ce
w at e r
j u c
Ae ro b act e r ae ro g e n e s
( = - . ++)
. . . . . . . . . . . 1 3 4 7 3 0 1 7
A. cl o acae ( - - ++)
. . . 3 4 2 3 1 4 3 1 2
Es che ri chi a co l i
( ++- - )
. . . . . . . . . . . . . 1 0 0 0 1 1
E. i n t e rm e d i a
( = F +- +) . . . . . . . . . . . . 0 1 3 0 0
U n cl as s i f i e d . . . . . . . . . . 0 0 8 3 1 4
*
V al u e s re p re s e n t t o t al n u m b e rs o f i s o l at e s .
t hat t he p re s e n ce o f E. co l i o n t he can e p l an t w as d u e t o
chan ce co n t am i n at i o n b y t he f e ce s o f b i rd s . In t he j u i ce
f ro m t he m i l l , ho w e v e r, E. co l i w as f o u n d t o b e p re s e n t at
t he hi g h d i l u t i o n s u s e d t o d e t e rm i n e t he n u m b e r o f A.
ae ro g e n e s an d A. cl o acae . T hi s co u l d b e a re f l e ct i o n o f t he
s o i l p i ck e d u p o n t he s u g arcan e af t e r cu t t i n g an d b e f o re
t ran s p o rt i n g t o t he s u g ar m i l l , o r o f t he can e j u i ce b e i n g a
f av o rab l e e n v i ro n m e n t f o r t he m u l t i p l i cat i o n o f t he E.
co l i t y p e .
T he o rg an i s m s l i s t e d as u n cl as s i f i e d had o n e o f t he
f o l l o w i n g IM\V i C co d e s : +- - +, +++- , - - +- ,
- + - - , an d - - - +. W i t h t he e x ce p t i o n o f t he t hre e
u n cl as s i f i e d o rg an i s m s f o u n d i n l e af - s he at h w at e r, al l t he
o rg an i s m s w i t h t he s e co d e s w e re f o u n d e i t he r i n can e
j u i ce o r o n t he b o t t o m p o rt i o n o f t he p l an t . T hi s s e e m s t o
i n d i cat e t hat t he u n cl as s i f i ab l e g ro u p o f co l i f o rm s i s a
re f l e ct i o n o f t he co l i f o rm t y p e s f o u n d i n t he s o i l rat he rt han
o f t he e p i p hy t i c t y p e s .
T he p re d o m i n at i n g IMV i C t y p e f o u n d o n t he can e p l an t ,
i n t he s he at hw at e r, an d i n t he can e j u i ce w as t he - - ++
t y p e ( T ab l e 8 ) . A t o t al o f 1 4 0 i s o l at e s b o re t hi s I1 \IV i C
co d e , charact e ri s t i c o f t he Ae ro b act e r g e n u s . T he n e x t
hi g he s t g ro u p w as co m p o s e d o f 2 0 i s o l at e s w i t h t he
+ - + + co d e , ag ai n re l at e d t o Ae ro b act e r. T he t y p i cal
Es che ri chi a co d e o f ++
- - had a t o t al o f 1 2 . M\Io re
v ari e t y am o n g t he IMV i C t y p e s s e e m e d t o o ccu r o n t he
can e p l an t t han i n t he o t he r t w o s o u rce s . Of a t o t al o f
1 0 8 i s o l at e s f ro m t he can e p l an t , 7 2 . 2 % w e re o f
t he - -
++ t y p e w i t h 1 4 . 8 % b e i n g o f t he +
-
++ t y p e .
Of a t o t al o f 3 6 i s o l at e s f ro m l e af - s he at h w at e r, 9 1 . 7 %
w e re o f t he - -
++ t y p e w i t h8 . 3 % b e i n g o f t he - + - -
t y p e . T he - +- - t y p e w as f o u n d o n l y i n t he s he at h
w at e r. Of a t o t al o f 5 4 i s o l at e s f ro m can e j u i ce , 5 3 . 7 %
w e re o f t he - - ++ t y p e , 2 0 . 4 % o f t he ++- - t y p e ,
an d 1 6 . 7 % o f t he - - - + t y p e , t he s e b e i n g t he p re -
d o m i n at e t y p e .
T he m aj o ri t y o f t he cu l t u re s o b t ai n e d f ro m s am p l e s o f
can e j u i ce i n cu b at e d at 4 5 . 5 C w e re o f t he Es che ri chi a
T ABLE 8 . Occu rre n ce o f d i f f e re n t IMV i C t y p e s o f co l i f o rm b act e ri a
i n 3 6 s am p l e s o f s u g arcan e p l an t s , 1 0 s am p l e s o f s u g arcan e j u i ce ,
an d 1 1 s am p l e s o f l e af - s he at h w at e r
IMV i C
re s u l t s
+++- -
7 +o t a
S o u rce o f i s o l at i o n
Can e p l an t S he at h w at e r Can e j u i ce
( 4 5 . 5 u c)
N o .
7 8
1
1 6
2
0
8
2
1
0
1 0 8
Pe r ce n t
7 2 . 2
0 . 9
1 4 . 8
1 . 9
7 . 4
1 . 9
0 . 9
N o .
3 3
0
0
0
3
0
0
0
0
3 6
Pe r ce n t
9 1 . 7
8 . 3
N o .
2 9
1 1
4
9
0
0
0
0
1
5 4
Pe r ce n t
5 3 . 7
2 0 . 4
7 . 4
1 6 . 7
1 . 8
N o .
6
1 8
0
0
0
0
0
0
0
2 4
Pe r ce n t
2 5 . 0
7 5 . 0
1 7 6 APPL. MICROBIOL.

o
n

S
e
p
t
e
m
b
e
r

1
9
,

2
0
1
4

b
y

g
u
e
s
t
h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m

COLIF ' ORM: IS AS S OCIAT ED W IT H S U GARCAN E
t y p e ; ho w e v e r, 2 5 . - ( , o f t he s e w e re al s o o f t he Ae r- o b act e r
t y p e .
T he re as o n f o i f i n d i n g o n l y P. ae ro g e n o i d e s an d n o t t y p i -
cal co l i f o rm o rg an i s m s o n y o u n g s u g arcan e p l an t s d u ri n g
t he e arl y p art o f t w o d i f f e re n t g ro w i n g s e as o n s i s n o t
u n d e rs t o o d .
It i s al s o f e as i b l e t hat t he co l i f o rm o rg an i s m s m ay g ai n
acce s s t o t he p l an t s as t he y o u n g p l an t s e m e rg e f ro m t he
g ro u n d . T he l ack o f m u l t i p l i cat i o n o f t he s e b act e ri a o n t he
y o u n g p l an t s m i g ht b e d u e t o t he v e ry s m al l q u an t i t y o f
n u t ri e n t m at e ri al s av ai l ab l e o n t he p l an t s u rf ace as co m -
p are d w i t h t he l arg e r q u an t i t y o f n u t ri e n t s s e e p i n g f ro m
t he s t al k s o f t he o l d e r p l an t s .
S i n ce t he d at a re p o rt e d he re i n d i cat e t hat t he ae ro g e n e s -
cl o acae t y p e s are re g u l arl y , rat he r t han i n t e rm i t t e n t l y ,
f o u n d o n o l d e r s u g arcan e p l an t s , i t ap p e ars t hat l arg e
n u m b e rs o f t he co l i f o rm s o n t he s e p l an t s are b y n o m e an s
acci d e n t al ; i t f o l l o w s t he n t hat t hi s e n v i ro n m e n t can b e
co n s i d e re d a n at u ral hab i t at w i t h t he o rg an i s m s b e i n g a
p art o f t he t ru e e p i p hy t i c m i cro f l o ra.
T he p re s e n ce o f l arg e n u m b e rs o f co l i f o rm o rg an i s m s i n
t he e x p re s s e d s u g arcan e j u i ce o f t he m i l l i s o f co n s i d e rab l e
i n t e re s t . T he cap ab i l i t y o f m e m b e rs o f t he g e n u s Ae ro -
b act e r t o p ro d u ce s l i m e w as re p o rt e d b y Be rk w i t h ( 1 9 3 1 )
an d S an b o rn ( 1 9 3 3 ) . I' re l i m i n ary i n v e s t i g at i o n s o n t he
e f f e ct o f p u re cu l t u re s o f Ae ro b act e r s p e ci e s o n s t e ri l e
s u g arcan e j u i ce s ho w e d t hat A. cl o acae i s cap ab l e o f p ro -
d u ci n g a l arg e am o u n t o f s l i m e i n t hi s m e n s t ru u m . Al -
t ho u g h L. m e s e n t e ro i d e s i s u s u al l y t ho u g ht o f as b e i n g t he
i m p o rt an t s l i m e - p ro d u ci n g o rg an i s m i n t he s u g ar m i l l ,
i t i s p o s s i b l e t hat t he Ae ro b act e r g ro u p he re t o f o re o v e r-
l o o k e d m i g ht b e q u i t e i m p o rt an t i n s l i m e p ro d u ct i o n an d
i n s u cro s e i n v e rs i o n . T he i m p o rt an ce o f f u rt he r s t u d i e s
o n t hi s g ro u p o f o rg an i s m s as re l at e d t o t he i r e f f e ct s i n
t he s u g ar m i l l can re ad i l y b e s e e n .
ACK N OW LEDGMEN T
T he f i n an ci al as s i s t an ce o f t he Lo u i s i an a S t at e U n i -
v e rs i t y F o u n d at i o n i s g rat e f u l l y ack n o w l e d g e d .
LIT ERAT U RE CIT ED
ALLEN , L. A. , AN D J . H ARRIS ON . 1 9 3 6 . T he charact e rs o f s o m e
co l i f o rm b act e ri a i s o l at e d f ro m g ras s an d g ras s s i l ag e . An n .
Ap p l . Bi o l . 2 3 : 5 3 8 - 5 4 5 .
AMERICAN PU BLIC H EALT H AS S OCIAT ION . 1 9 5 5 . S t an d ard m e t ho d s
f o r t he e x am i n at i o n o f w at e r, s e w ag e , an d i n d u s t ri al w as t e s ,
1 0 t h e d . Am e ri can Pu b l i c H e al t h As s o ci at i o n , N e w Yo rk .
BERK W IT H , T . D. 1 9 3 1 . T he b act e ri o l o g y o f p u l p s l i m e . J . Bact e -
ri o l . 2 2 : 1 5 - 2 2 .
F IS H BEIN , M. 1 9 6 2 . T he ae ro g e n i c re s p o n s e o f Es che ri chi co l i an d
s t rai n s o f Ae ro b act e r i n EC b ro t h- an d s e l e ct e d s u g ar b ro t hs at
e l e v at e d t e m p e rat u re s . Ap p l . Mi cro b i o l . 1 0 : 7 9 - 8 5 .
F RAS ER, M. H . , W . B. REID, AN D J . F . M. ALCOLM. 1 9 5 6 . T he o c-
cu rre n ce o f co l i - ae ro g e n e s o rg an i s m s o n p l an t s . J . Ap p l .
Bact e ri o l . 1 9 : 3 0 1 - 3 0 9 .
GEERLIGS , P. 1 9 2 4 . Can e s u g ar an d i t s m an u f act u re , 2 n d e d .
N o rm al Ro g e r, Lo n d o n .
GELDREICH , E. E. , C. B. H U F F , AN D L. C. BES T . 1 9 6 3 . T y p e d i s -
t ri b u t i o n o f co l i f o rm b act e ri a o n v e g e t at i o n . Bact e ri o l . Pro c. ,
p . 1 1 .
H AJ N A, A. A. , AN D S . R. DAMON . 1 9 3 4 . Di f f e re n t i at i o n o f A. ae ro -
g e n e s an d A. cl o acae o n t he b as i s o f t he hy d ro l y s i s o f s o d i u m
hi p p u rat e . Am . J . H y g . 1 9 : 5 4 5 - 5 4 8 .
H AYN ES , W . C. , L. J . W ICK ERH AM, AN D C. W . H ES S ELT IN E. 1 9 5 5 .
Mai n t e n an ce o f cu l t u re s o f i n d u s t ri al l y i m p o rt an t m i cro o r-
g an i s m s . Ap p l . Mi cro b i o l . 3 : 3 6 1 - 3 6 8 .
H IJ T CH IS ON , C. M. , AN D C. S . RAMAYYAR. 1 9 2 5 . Lo s s o f s u g ar b y
i n v e rs i o n i n s u g ar f act o ri e s i n n o rt he rn In d i a an d i t s p re v e n -
t i o n b y an t i s e p t i c m e as u re s . Ag ri c. Re s . In s t . Pu s a, In d i a,
Bu l l . N o . 1 6 3 , p . 9 .
K OPELOF F , N . , AN D L. K OPELOF F . 1 9 1 9 . T he d e t e ri o rat i o n o f can e
s u g ar b y f u n g i . Lo u i s i an a Ag r. Ex p t . S t a. Bu l l . 1 6 6 , p . 1 - 7 2 .
K ROU LIK , J . T . , L. A. BU RK EY, AN D H . G. W IS EMAN . 1 9 5 5 . T he
m i cro b i al p o p u l at i o n s o f t he g re e n p l an t an d o f t he cu t f o rag e
p ri o r t o e n s i l i n g . J . Dai ry S ci . 3 8 : 2 5 6 - 2 6 2 .
MAYEU X, P. A. 1 9 6 0 . S o m e s t u d i e s o n t he m i cro b i al f l o ra o f s u g ar-
can e . M. S . T he s i s , Lo u i s i an a S t at e U n i v e rs i t y , Bat o n Ro u g e .
MILLS T EIN , C. H . , L. T OBIN , AN D C. S . MCCLES K EY. 1 9 4 1 . A b ac-
t e ri o l o g i cal s t u d y o f t he m an u f act u re o f raw can e s u g ar. S u g ar
J . 3 : 1 3 - 1 4 .
OW EN , W . L. 1 9 1 1 . A re ce n t l y d i s co v e re d b act e ri al d e co m p o s i t i o n
o f s u cro s e . J . In d . En g . Che m . 3 : 4 8 1 - 4 8 6 .
PEDERS ON , C. S . 1 9 3 8 . A b act e ri o l o g i cal s t u d y o f raw can e s u g ar
p l an t s . J . Bact e ri o l . 3 5 : 7 4 - 7 5 .
S AN BORN , J . R. 1 9 3 3 . De v e l o p m e n t an d co n t ro l o f m i cro o rg an i s m s
i n a p u l p an d p ap e r m i l l s y s t e m . J . Bact e ri o l . 2 6 : 3 7 3 - 3 7 8 .
T H OMAS , S . B. , AN D P. M. H OBS ON . 1 9 5 5 . Co l i - ae ro g e n e s b act e ri a
i s o l at e d f ro m e ars an d p an i cl e s o f ce re al cro p s . J . Ap p l . Bac-
t e ri o l . 1 8 : 1 - 8 .
T H OMAS , S . B. , AN D J . MCQ U ILLIN . 1 9 5 2 . Co l i - ae ro g e n e s b act e ri a
i s o l at e d f ro m g ras s . Pro c. S o c. Ap p l . Bact e ri o l . 1 5 : 4 1 - 5 2 .
W OLZOGEN K U H R, C. A. H . 1 9 2 3 . In v e s t i g at i o n s o n t he m i ro f l o ra
o f n o rm al s u g arcan e an d o f can e af f e ct e d b y s e re h d i s e as e .
Arch. S u i k e ri n d . N e d . In d i e I Me d e d e e l . Pro e f s t a. J av a-
S u i k e ri n d . N o . 9 , p . 3 2 1 - 4 8 4 .
V OL. 1 2 , 1 9 6 4
1 7 7

o
n

S
e
p
t
e
m
b
e
r

1
9
,

2
0
1
4

b
y

g
u
e
s
t
h
t
t
p
:
/
/
a
e
m
.
a
s
m
.
o
r
g
/
D
o
w
n
l
o
a
d
e
d

f
r
o
m