A method for the detection of bacteriophages from ocean water

H E L G O ~ D E R MEERESUNTERSUCHUNGEN Helgol~inder Meeresunters. 34, 1-14 {1980)

A m e t h o d f o r t h e d e t e c t i o n of b a c t e r i o p h a g e s f r o m

o c e a n w a t e r

K. Moebus

Biologische Anstalt Helgoland (Meeresstation); D-2192 Helgoland,

Federal Republic of Germany

ABSTRACT: A method for the isolation of bacteriophages from ocean water is described. It precludes sample storage before starting phage-enrichment cultures and provides for the use of 3 sub-samples enriched with organic nutrients after 1, 2 and 3 days of incubation, The method was used with samples collected from 6 m below the surface at 48 stations between the European continental shelf and the Sargasso Sea. With 213 among 931 bacterial isolates about 250 strains of bacteriophages were detected by two methods of different sensitivity. From 14 samples taken east of the Azores 115 host bacteria have been found versus only 98 from 34 samples collected at westerly stations, The employment of more than one sub-sample per station as well as the use of more sensitive phage-detection procedures was found to be more advantageous the lower the concentra- tion of cultivatable bacteria in a sample.

I N T R O D U C T I O N

A m o n g p r e v i o u s l y d e s c r i b e d m e t h o d s for t h e i s o l a t i o n of b a c t e r i o p h a g e s from s e a w a t e r {Spencer, 1955, 1960; H i d a k a , 1971, 1977) o n l y t h a t of H i d a k a {1977) is s u i t a b l e for c o n d i t i o n s a b o a r d ship. T h i s m e t h o d i n v o l v e s s t a r t i n g t h e c u l t u r a l e n r i c h m e n t of p h a g e s b e f o r e host b a c t e r i a a r e a v a i l a b l e , so t h a t s t o r a g e of w a t e r s a m p l e s for a w e e k or e v e n l o n g e r is a v o i d e d . H i d a k a ' s m e t h o d , h o w e v e r , n e i t h e r e x c l u d e s s a m p l e s t o r a g e for at l e a s t o n e d a y nor e l i m i n a t e s t h e n e e d of c o n s i d e r a b l e s p a c e in a c o o l e d r o o m to store the t r e a t e d s a m p l e s u n t i l return. T h e r e f o r e , it c a n b e e m p l o y e d o n l y d u r i n g r e l a t i v e l y short cruises.

W h e n p a r t i c i p a t i n g in a n 1 1 - w e e k e e l b i o l o g y e x p e d i t i o n to t h e S a r g a s s o S e a w i t h the 370 ton r e s e a r c h v e s s e l " F r i e d r i c h H e i n c k e " of t h e B i o l o g i s c h e A n s t a l t H e l g o l a n d , I r e a l i s e d t h e n e c e s s i t y of a m e t h o d r e q u i r i n g t h e l e a s t p o s s i b l e s t o r a g e room.

W i t h s e a w a t e r c o l l e c t e d n e a r H e l g o l a n d a n e n r i c h m e n t c u l t u r e m e t h o d p r e c l u d i n g s a m p l e s t o r a g e w a s d e v e l o p e d a n d t e s t e d w i t h o c e a n w a t e r d u r i n g a c r u i s e to t h e Bay of Biscay in O c t o b e r 1978. A f t e r m i n o r a d j u s t m e n t s t h e p r o c e d u r e , w h i c h w i l l b e d e s c r i b e d a n d e v a l u a t e d in this p a p e r , w a s u s e d d u r i n g cruise no. 160 of RV " F r i e d r i c h H e i n c k e " lasting from F e b r u a r y 12 t h r o u g h A p r i l 26, 1979.

2 K. M o e b u s

MATERIALS A N D M E T H O D S M e d i a

S e a w a t e r a g a r {SWA), soft s e a w a t e r a g a r (sSWA) a n d s e a w a t e r b r o t h (SWB/5) w e r e p r e p a r e d w i t h 75% a g e d North Sea w a t e r a n d 25 % distilled water. S W A a n d sSWA c o n t a i n e d 5 g p e p t o n e (Difco), 1 g y e a s t extract (Difco}, a n d 0.1 g FePO4 p e r liter b u t differed i n t h e i r a g a r (Difco} c o n t e n t w h i c h w a s 1.5 % a n d 0.6 %, r e s p e c t i v e l y . SWB/5 w a s p r e p a r e d w i t h 1 g p e p t o n e (Difco), 0.2 g y e a s t extract (Difco), a n d 0.1 g FePO 4 p e r liter. T h e p H of t h e s e m e d i a w a s a b o u t 7.6 after a u t o c l a v i n g for 20 m i n at 121 ° C.

C o n c e n t r a t e d p e p t o n e - y e a s t extract s o l u t i o n (25 × PY) c o n t a i n i n g 125 g p e p t o n e (Difco) a n d 25 g y e a s t extract (Difco} p e r liter, a n d a 3 % s o l u t i o n of Difco beef extract (BES) w e r e p r e p a r e d w i t h d i s t i l l e d w a t e r only.

T h e m e d i a w e r e d i s p e n s e d into s c r e w - c a p b o t t l e s or t u b e s i n a m o u n t s c o r r e s p o n d i n g to the e x p e c t e d d e m a n d p e r s e a w a t e r s a m p l e to b e i n v e s t i g a t e d . In addition, 1400 Petri d i s h e s c o n t a i n i n g 20 ml SWA e a c h w e r e p r e p a r e d a n d s e a l e d i n sterile p l a s t i c b a g s i m m e d i a t e l y after c o o l i n g to room t e m p e r a t u r e . All m e d i a w e r e stored i n the d a r k at 5 °C.

S a m p l i n g of s e a w a t e r

S e a w a t e r s a m p l e s w e r e c o l l e c t e d a s e p t i c a l l y from 6 m b e l o w the surface (3 m b e l o w the keel) w i t h a s a m p l e r h o l d i n g 2 bottles of 5- a n d 1-1 capacity, w h i c h w e r e c o n n e c t e d to a c o m m o n i n l e t tube. S a m p l i n g g e n e r a l l y took less t h a n 5 min.

T r e a t m e n t of s e a w a t e r s a m p l e s

All s a m p l e s w e r e t r e a t e d i m m e d i a t e l y after c o l l e c t i o n to e n r i c h b a c t e r i o p h a g e s a n d to c u l t i v a t e b a c t e r i a .

Enrichment of bacteriophages

T h r e e s u b - s a m p l e s of 1.5 1 s e a w a t e r w e r e d i s p e n s e d into sterile bottles of 2-1 capacity. At zero t i m e 12 m l of 25 × PY w e r e a d d e d to S u b - s a m p l e 1 r e s u l t i n g i n a c o n c e n t r a t i o n of p e p t o n e a n d y e a s t extract c o r r e s p o n d i n g to that i n SWB/5. S u b - s a m p l e s 2 a n d 3 r e c e i v e d t h e s a m e a m o u n t s of n u t r i e n t s o n l y after 24 a n d 48 h of i n c u b a t i o n , r e s p e c t i v e l y ( p r e - i n c u b a t e d s u b - s a m p l e s ) . I n c u b a t i o n w a s n o r m a l l y p e r f o r m e d i n still c u l t u r e at 20 ° to 21 °C, b u t for s e v e r a l w e e k s at a m b i e n t t e m p e r a t u r e (23 ° to 26°C) d u e to a b r e a k d o w n of t h e c o o l i n g e q u i p m e n t .

From e a c h s u b - s a m p l e a l i q u o t s of 10 m l w e r e t a k e n 1, 2 a n d 3 days after the a d d i t i o n of 25 × PY a n d c e n t r i f u g e d for 20 m i n at a b o u t 2000 r p m to r e m o v e most of the p r o f u s e l y g r o w n b a c t e r i a . C e n t r i f u g a t i o n w a s p e r f o r m e d o n a t a b l e s u s p e n d e d b y a c a r d a n joint. F i n a l l y the s u p e r n a t a n t s w e r e filtered t h r o u g h 0.15-/zm c e l l u l o s e - n i t r a t e filters (Sarto- rius, G 6 t t i n g e n ) p r e v i o u s l y w a s h e d w i t h 1 m l BES to p r e v e n t p h a g e adsorption.

B a c t e r i o p h a g e s from o c e a n w a t e r 3 syringes a t t a c h e d to t h e filter holders. T h e b o x itself w a s affixed o n t h e t a b l e b y m e a n s of d o u b l e - s i d e d sticky tape. T h e n BES w a s s l o w l y p a s s e d t h r o u g h t h e filters a n d the s u p e r n a t a n t s p o u r e d i n t o t h e s y r i n g e bodies. T h e f i l t e r - s y r i n g e a s s e m b l i e s w e r e r e m o - ved from the box o n e after the other a n d the s u p e m a t a n t s filtered d i r e c t l y into sterile plastic s c r e w - c a p t u b e s to b e stored at 5 °C.

In this w a y 9 p r e p a r a t i o n s w e r e o b t a i n e d from e a c h s e a w a t e r s a m p l e to b e c h e c k e d for the p r e s e n c e of b a c t e r i o p h a g e s .

Cultivation o f bacteria

Bacterial colonies w e r e g r o w n o n SWA i n o c u l a t e d w i t h 0.1 m l p e r p l a t e a n d i n c u b a t e d for 7 days at 25 °C. Before u s e the SWA p l a t e s w e r e k e p t at r o o m t e m p e r a t u r e (usually a b o v e 20 °C) for 2 to 3 days to a t t a i n t h e d e s i r e d d e g r e e of d r y n e s s a n d to s h o w possible c o n t a m i n a t i o n as well.

Since the n u m b e r of c o l o n y - f o r m i n g u n i t s (CFU) is g e n e r a l l y very l o w i n o c e a n w a t e r the f o l l o w i n g p r o c e d u r e w a s e m p l o y e d w i t h all s e a w a t e r s a m p l e s i n v e s t i g a t e d to c o n c e n t r a t e b a c t e r i a before c u l t i v a t i o n . U s i n g s t e r i l i z e d e q u i p m e n t only, s e a w a t e r w a s d r a w n b y v a c u u m from a g r a d u a t e d c y l i n d e r i n t o a filter a p p a r a t u s t h r o u g h t u b i n g r u n n i n g from the t i g h t l y fitting lid of t h e filter f u n n e l to t h e b o t t o m of the c y l i n d e r . T h e latter was closed w i t h a lid b e a r i n g a hole for the t u b i n g . N u c l e p o r e ® filters of 47 m m d i a m e t e r a n d 0 . 2 / a n pore size w e r e u s e d to collect s u s p e n d e d m a t e r i a l .

T h e g r e a t e r part of the r e t a i n e d b a c t e r i a w a s w a s h e d off t h e filter b y r e p e a t e d s q u i r t i n g of t h e s a m e 2 to 3 m l of s e a w a t e r w i t h a p i p e t t e . F i n a l l y , t h e s u s p e n s i o n w a s transferred to a t u b e a n d s p r e a d o n series of 5 SWA p l a t e s e a c h u n d i l u t e d as w e l l as after 2-, 5-, a n d 10-fold d i l u t i o n .

T h e filter w a s p l a c e d o n SWA, e x a m i n e d u n d e r a d i s s e c t i n g microscope, a n d i n c u b a t e d for 7 days at 25 °C. T h e a m o u n t of s e a w a t e r filtered a n d the t i m e n e e d e d for this process w e r e recorded.

From a few s a m p l e s only, u n t r e a t e d s e a w a t e r w a s also s p r e a d o n SWA. S i n c e the n u m b e r of colonies w a s a l w a y s v e r y l o w this m e t h o d w a s a b a n d o n e d . H o w e v e r , w i t h all b u t 3 s a m p l e s i n v e s t i g a t e d the n u m b e r of C F U w a s d e t e r m i n e d i n t h e 2 p r e - i n c u b a t e d s u b - s a m p l e s just prior to t h e a d d i t i o n of 25 x PY.

After i n c u b a t i o n , as m a n y s e e m i n g l y different c o l o n i e s (or parts thereof) as p o s s i b l e w e r e p i c k e d from the plates. P r e f e r e n t i a l l y the c o l o n i e s w e r e s e l e c t e d from S W A i n o c u l a t e d w i t h t h e c o n c e n t r a t e of p a r t i c u l a t e matter. Since, h o w e v e r , often less t h a n the m i n i m u m n u m b e r of 30 different c o l o n i e s c o u l d b e f o u n d o n t h e s e plates, for most of the w a t e r s a m p l e s c o l o n i e s w e r e also p i c k e d from S W A i n o c u l a t e d w i t h m a t e r i a l of the pre- i n c u b a t e d s u b - s a m p l e s .

T h e s e l e c t e d m i c r o b e s w e r e t r a n s f e r r e d into 2 m l SWB/5 a n d i n c u b a t e d for 2 days at 25 °C. T h e s e c u l t u r e s t h e n w e r e s t r e a k e d o n S W A s l a n t s w h i c h , after i n c u b a t i o n for a n o t h e r 2 days at the s a m e t e m p e r a t u r e , w e r e stored at 5 °C.

D e t e c t i o n of b a c t e r i o p h a g e s

4 K. M o e b u s

e m p l o y i n g p l a t e s with 10 ml of SWA as bottom l a y e r and 2.5 ml of sSWA as top layer, the latter s e e d e d with bacteria.

At first a common spot test was p e r f o r m e d with small amounts of the p r e p a r a t i o n s transferred onto the top layers b y m e a n s of a multi-point inoculator. This test r e v e a l e d b a c t e r i o p h a g e s only if t h e y w e r e p r e s e n t in fairly l a r g e numbers. To d e t e c t rare phages, a n o t h e r series of e x p e r i m e n t s was p e r f o r m e d e m p l o y i n g so-called ring holders.

The ring h o l d e r s w e r e p r o d u c e d in the institute's workshop from Lexan ® sheet (3 m m thick) a n d M a k r o l o n ® rings (8 m m high, 15 m m outer diameter), the latter g l u e d into holes d r i l l e d in the sheet. They can b e s t e r i l i z e d by autoclaving (20 min at 121 °C).

D o u b l e - l a y e r p l a t e s w e r e p o u r e d as in the a f o r e - m e n t i o n e d test. This time, however, only those b a c t e r i a w e r e u s e d w h i c h h a d not b e e n found to be suitable hosts during the first test series. In e a c h p l a t e a ring h o l d e r was p r e s s e d right through both a g a r layers t h e r e b y forming 9 wells, into w h i c h 0.1 ml of the p r e p a r a t i o n s was p l a c e d . The plates w e r e i n c u b a t e d overnight at 25 °C. After i n c u b a t i o n some liquid was w i t h d r a w n from the w e l l s to b e s p o t - t e s t e d on freshly p o u r e d top layers s e e d e d with the respective bacteria. This p r o c e d u r e w a s n e c e s s a r y to o b t a i n clear-cut results w h i c h h a d not b e e n p r o d u c e d with the p l a t e s c o n t a i n i n g the ring holders. With the ring holders in p l a c e or a l r e a d y removed, only rarely w e r e u n e q u i v o c a l i n d i c a t i o n s of lysis observed, w h e r e a s with the l i q u i d from m a n y unsuspicious wells strong lyric action was found after re-testing. Each lysis-positive spot was r e m o v e d from the p l a t e a n d transferred into 2.5 ml of SWB/5 to be stored as e l u a t e in the refrigerator.

I s o l a t i o n a n d p u r i f i c a t i o n of b a c t e r i o p h a g e s

To isolate the d e t e c t e d p h a g e s , serial dilutions of either the p r e p a r a t i o n s p r o d u c e d from s u b - s a m p l e s or the e l u a t e s m e n t i o n e d in the last p a r a g r a p h w e r e u s e d to pour d o u b l e - l a y e r p l a t e s with the r e s p e c t i v e host bacteria. Generally, with each strain of host b a c t e r i a only one r e p r e s e n t a t i v e p r e p a r a t i o n or e l u a t e p e r s u b - s a m p l e was titrated. Only if the p r e s e n c e of different p h a g e s was i n d i c a t e d b y the results of the spot tests d e s c r i b e d above was more than one p r e p a r a t i o n or e l u a t e used. After i n c u b a t i o n at 25 °C single p l a q u e s w e r e i s o l a t e d from the p l a t e s a n d e l u t e d in 1 ml of s e a w a t e r each. These eluates a g a i n w e r e titrated with the r e s p e c t i v e host b a c t e r i a to p r o d u c e another g e n e r a t i o n of single plaques. To purify the i s o l a t e d p h a g e s , this p r o c e d u r e was r e p e a t e d at least three times.

During the process of isolation a n d purification, d o u b l e t s of p h a g e s and host b a c t e r i a d e r i v e d from the same s a m p l e of s e a w a t e r (intrasample doublets) were e l i m i n a - ted. The d e s i g n a t i o n of b a c t e r i o p h a g e s as d o u b l e t s was g e n e r a l l y b a s e d on the similarity of a p p e a r a n c e of the p l a q u e s they p r o d u c e d with a b a c t e r i a l host. Concerning host bacteria, d o u b l e t s w e r e d e t e c t e d b y spot t e s t i n g all r e m a i n i n g isolates of b a c t e r i o p h a g e s on top layers s e e d e d with the host b a c t e r i a i s o l a t e d from the same s e a w a t e r sample. This h o s t - p h a g e cross infection test was p e r f o r m e d after 3 consecutive isolations of single plaques.

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6 K . M o e b u s

T a b l e 1. P h y s i c a l d a t a o f m i c r o b i o l o g i c a l s t a t i o n s

S t a t i o n P o s i t i o n D a t e T i m e o f s a m p l i n g T e m p e r a t u r e (°C) n o . L a t i t u d e L o n g i t u d e S h i p G M T W a t e r A i r

4 5 4 4 7 ° 4 3 , N 0 6 ° 3 8 , W F e b r u a r y 15 0 7 : 3 0 = 1 1 . 5 3 . 0 4 5 6 4 4 ° 4 5 , N 10055 , W 1 6 0 8 : 2 0 = 1 4 . 5 8 . 0

4 5 8 4 3 ° 1 2 , N 14017 , W 17 0 9 : 1 0 = 1 5 . 0 13.0

B a c t e r i o p h a g e s from o c e a n w a t e r 7

RESULTS

S e a w a t e r s a m p l e s w e r e c o l l e c t e d at t h e 48 s t a t i o n s s h o w n i n F i g u r e 1. A d d i t i o n a l information is c o m p i l e d in T a b l e 1. T h e d e p t h at t h e s a m p l i n g s i t e s w a s b e t w e e n s e v e r a l h u n d r e d a n d s e v e r a l t h o u s a n d m e t r e s w i t h t h e f o l l o w i n g e x c e p t i o n : 120 m a n d 144 m at Station nos. 454 a n d 605, r e s p e c t i v e l y (Bay of Biscay), 60 m at S t a t i o n no. 489 (Plan- t a g e n e t Bank, s o u t h w e s t of B e r m u d a ) a n d 12 m at S t a t i o n no. 565 ( a b o u t 800 m off St. G e o r g e ' s , Bermuda).

For t h e b a c t e r i a t h e m a i n p u r p o s e of t h e e x p e r i m e n t s p e r f o r m e d w a s to i s o l a t e a r e a s o n a b l y l a r g e n u m b e r of d i f f e r e n t b a c t e r i a l s t r a i n s b u t n o t n e c e s s a r i l y to c o l l e c t e x a c t d a t a on the b a c t e r i a l c o n t e n t of t h e s e a w a t e r s a m p l e s . T h e r e f o r e , o n l y r o u g h e s t i m a t e s of the n u m b e r s of C F U w e r e o b t a i n e d ( T a b l e 2). T h e y a r e b a s e d on t h e n u m b e r s of CFU ml 1 f o u n d w i t h the s u s p e n s i o n s of c o n c e n t r a t e d p a r t i c u l a t e m a t e r i a l a n d t h e e s t i m a t e d n u m b e r s of c o l o n i e s g r o w n on t h e filters u s e d in t h e c o n c e n t r a t i o n p r o c e d u r e , t a k i n g into a c c o u n t the r e s p e c t i v e c o n c e n t r a t i o n factor. T a b l e 2 a l s o p r e s e n t s t h e n u m - b e r s of CFU m l "~ d e t e r m i n e d for s u b - s a m p l e s p r e - i n c u b a t e d w i t h o u t o r g a n i c n u t r i e n t s a d d e d . In most c a s e s the r e s u l t s o b t a i n e d w i t h r e l a t e d s u b - s a m p l e s after 24 h of p r e - i n c u b a t i o n a g r e e f a i r l y well. M o s t l y t h e y a r e also r e a s o n a b l y c o r r e l a t e d w i t h t h e r o u g h e s t i m a t e s of CFU p r e s e n t in t h e s e a w a t e r at c o l l e c t i o n t i m e , e s p e c i a l l y w h e n t h e s e a r e v e r y low. T h e r e f o r e , it is s u p p o s e d t h a t t h e r o u g h e s t i m a t e s of t h e CFU c o n t e n t of t h e s a m p l e s p r e s e n t e d in T a b l e 2 a r e n o t u n r e a l i s t i c . H o w e v e r , t h e y m a y b e s o m e w h a t too tow s i n c e N u c l e p o r e ® filters of 0.2-~m p o r e s i z e d o not r e t a i n a l l b a c t e r i a .

At the e n d of t h e c r u i s e 1382 m i c r o b i a l i s l a t e s h a d b e e n c o l l e c t e d , 451 of w h i c h d i d not s u r v i v e u n t i l t h e y c o u l d b e u s e d to d e t e c t b a c t e r i o p h a g e s in t h e p r e p a r a t i o n s m a d e from p h a g e - e n r i c h m e n t cultures. A m o n g t h e r e m a i n i n g 931 i s o l a t e s , 213 w e r e f o u n d to b e s u i t a b l e hosts for b a c t e r i o p h a g e s , i n c l u d i n g 36 d o u b l e t s .

T h e d i s t r i b u t i o n of host b a c t e r i a a m o n g a l l m i c r o b i a l s t r a i n s i s o l a t e d from t h e 48 s e a w a t e r s a m p l e s is s h o w n in F i g u r e 2. W i t h 70 % of t h e hosts, b a c t e r i o p h a g e s w e r e d e t e c t e d d u r i n g the first t e s t s e r i e s e m p l o y i n g t h e m u l t i - p o i n t i n o c u l a t o r ( s q u a r e s i n Fig. 2), w h e r e a s w i t h the r e m a i n i n g host b a c t e r i a (circles in Fig. 2) p h a g e s c o u l d b e f o u n d o n l y in t h e s e c o n d , m o r e s e n s i t i v e test s e r i e s u s i n g t h e r i n g h o l d e r s . As r e v e a l e d d u r i n g the i s o l a t i o n p r o c e d u r e , b a c t e r i o p h a g e s d e t e c t e d b y m e a n s of t h e m u l t i - p o i n t i n o c u l a t o r w e r e p r e s e n t in t h e p r e p a r a t i o n s at n u m b e r s r a n g i n g from a b o u t 10 m1-1 to 5 × 10 ~ m l 1. W i t h 40 % of t h e h o s t b a c t e r i a , b a c t e r i o p h a g e s w e r e f o u n d in p r e p a r a t i o n s of a l l 3 r e l a t e d s u b - s a m p l e s , w h e r e a s w i t h a b o u t 35 % of t h e hosts, b a c t e r i o p h a g e s w e r e d e t e c t e d in p r e p a r a t i o n s of o n l y o n e of t h e 3 s u b - s a m p l e s . T a b l e 3 p r e s e n t s t h e s e f i n d i n g s in d e t a i l . In T a b l e 4 t h e n u m b e r s of h o s t b a c t e r i a a r e g i v e n w i t h w h i c h b a c t e r i o p h a g e s w o u l d h a v e b e e n f o u n d w i t h 1, 2 or 3 s u b - s a m p l e s on t h e o n e h a n d a n d , on the o t h e r h a n d , if p r e p a r a t i o n s w o u l d h a v e b e e n p r o d u c e d o n l y after 1, 2, I a n d 2, or 2 a n d 3 d a y s of i n c u b a t i o n after n u t r i e n t e n r i c h m e n t of t h e s u b - s a m p l e s .

Table 2 (Continued) Station no. Number of CFU ml -i Concentration in concentrate of factor particulate matter Calculated number of CFU ml -I in sample (see text) 531 2.3 x 102 461 ~ 1 536 2.8 x 102 467 ~ 2 541 7.3 x 102 493 ~ 3 547 1.6 x 102 361 ~ 2 554 2,6 X 102 316 ~ 2 559 3.1 X 102 431 ~ 2 564 1,6 X 103 337 ~10 565 1.5 X 104 388 >40 568 1.3 X 103 345 ~ 8 570 2,1 X 103 279 ~15 572 6.7 X 102 246 ~ 5 576 4,8 X 103 325 ~20 581 2.3 X 103 287 >10 588 4.1 X 102 345 ~ 2 590 1,6 X 104 336 >50 593 3.8 X 103 366 ~15 596 5.0 X 103 415 >20 598 4.8 x 103 417 >20 600 2.1 X 104 353 >60 601 4.4 X 103 240 >30 602 1.9 X 103 364 >10 603 2.3 X 103 351 ~12 605 9.0 X 102 347 -- 8 Number of CFU m1-1 in sub-sample 2 sub-sample 3 after pre-incubation for 24 h 24 h 48 h 101 101 >102 2 x l0 t 3 × 101 103 >102 5 x 10 Z 6 X 103 2 × l0 t >101 8 X 102 2 x 10 t 2 x lO I 3 X 102 >10t >101 5 X 10 2 3,8 x 102 102 1,5 X 103 5.8 x 102 3 x 102 4 x 102 1.8 X 103 ~4 X 103 1.2 X 104 2,6 x 102 2,6 x 102 1.2 × 103 102 1.3 x 102 1.7 X 103 1.6 × 102 1.4 × 102 3.8 X 103 9 X I0 ~ 9 x 10 ~ 1.2 × 103 <10 t 2 x l0 t 10 3 ~7 × 10 2 2.6 x 10 3 5 x l0 4 n.d. 2 x 102 2 x 103 1,2 x 103 >2 × 103 ~5 x 103 5.6 x 102 6 × 102 >3 × 104 1.7 x 102 2.4 x 102 2.3 X 103 102 1,6 x 102 1.5 X 103 6 x 102 2,5 x 102 2.5 X 103 1.2 x 103 1.9 X 103 1.2 X 104 n.d. 3.2 X 102 3.5 X 103 Incubation (°C)

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Fig. 2. M i c r o b i a l i s o l a t e s a s c o l l e c t e d f r o m 48 A t l a n t i c O c e a n w a t e r s a m p l e s , P l a i n a r e a s : i s o l a t e s d e r i v e d f r o m c o n c e n t r a t e of p a r t i c u l a t e m a t t e r ; d a s h e d a r e a s : i s o l a t e s o b t a i n e d f r o m p r e - i n c u b a t e d s u b - s a m p l e s ; b a r s i n d i c a t e lost i s o l a t e s ; • + [] : h o s t b a c t e r i a f o u n d d u r i n g p h a g e - d e t e c t i o n t e s t s e m p l o y i n g m u l t i - p o i n t i n o c u l a t o r ; • + © : h o s t b a c t e r i a d e t e c t e d i n t e s t s u s i n g ring h o l d e r s ; o p e n

s y m b o l s : b a c t e r i a l d o u b l e t s a m o n g i s o l a t e s of r e s p e c t i v e s a m p l e

B a c t e r i o p h a g e s f r o m o c e a n w a t e r 11 Table 3. N u m b e r s of host b a c t e r i a i s o l a t e d from 48 o c e a n i c w a t e r s a m p l e s in r e l a t i o n to the detection of b a c t e r i o p h a g e s from 3 r e l a t e d s u b - s a m p l e s p e r s a m p l e b y two p h a g e - d e t e c t i o n

p r o c e d u r e s

Host b a c t e r i a i n d i c a t i n g the p r e s e n c e of

In s u b - s a m p l e s b a c t e r i o p h a g e s i n test series e m p l o y i n g S u m m u l t i - p o i n t i n o c u l a t o r r i n g h o l d e r s

1 to 3 71 (48.0 %) 15 (23,1%) 86 (40.4 %)

1 only 18 (12.2 %) 16 (24,6 %) 34 (16.0 %)

1 + 2 17 (11.5%} 7 (10.8 %) 24 {11.3%)

1 + 3 7 (4.7 %) 6 (9.2 %) 13 ( 6 . 1 % )

2 only 8 (5.4 %) 5 (7.7 %) 13 (6.1%)

2 + 3 10 (6.7 %) 5 (7.7 %) 15 (7.0 %)

3 only 17 (11.5 %) 11 (16.9 %) 28 (13.1%)

148 (100 %) 65 (100 %) 213 (100 %)

T a b l e 4. N u m b e r s of host b a c t e r i a isolated from 48 o c e a n i c w a t e r s a m p l e s i n r e l a t i o n to t h e detection of b a c t e r i o p h a g e s w i t h p r e p a r a t i o n s d e r i v e d from r e l a t e d s u b - s a m p l e s . T h e i r d e p e n d e n c e u p o n n u m b e r of s u b - s a m p l e s p e r s a m p l e a n d s c h e d u l e of p r e p a r a t i o n production. 213 host b a c t e r i a

= 100 % S u b - s a m p l e s

i n c u b a t e d (days)

Host b a c t e r i a i n d i c a t i n g the p r e s e n c e of b a c t e r i o p h a g e s in p r e p a r a t i o n s m a d e from

S u b - s a m p l e 1 only S u b - s a m p l e s 1 a n d 2 S u b - s a m p l e s 1 to 3

1 90 (42.3 %) 123 (57.8 %) 149 (70.0 %)

2 144 (67.6 %) 168 (78,9 %) 194 (91.1%)

1 + 2 149 (70.0 %) 175 (82.2 %) 202 (94.9 %)

2 + 3 152 (71.4 %) 179 (84.0 %) 206 (96.7 %)

T a b l e 5. N u m b e r s of collected a n d lost m i c r o b i a l isolates in r e l a t i o n to source a n d p i g m e n t p r o d u c t i o n

Source

M i c r o b i a l C o l o u r e d M i c r o b i a l C o l o u r e d

isolates isolates isolates isolates

collected collected lost lost

Particulate m a t t e r con- 1083 276 3 i 0 114

centrate of sea w a t e r (100 %) (100 %) (28.6 %) (41.4 %)

P r e - i n c u b a t e d sub- 299 93 141 80

samples 2 a n d 3 (100 %) (100 %) (47.2 %) (86.0 %)

12 K. M o e b u s

i s o l a t e s t h e p e r i o d of t i m e t h e y r e m a i n v i t a l on S W A slants, w h i c h is o b v i o u s l y i n d e p e n - d e n t of t h e i r a b i l i t y to g r o w on t h i s m e d i u m , g e n e r a l l y s e e m s to b e s h o r t e r t h a n that o b s e r v e d w i t h t h e m a j o r i t y of b a c t e r i a i s o l a t e d from N o r t h S e a w a t e r ,

T h e s o u r c e of t h e i s o l a t e s a n d t h e i r a b i l i t y to form p i g m e n t s w e r e o b s e r v e d to affect s u r v i v a l . T h e d a t a p r e s e n t e d in T a b l e 5 s h o w t h a t t h e p o r t i o n of lost m i c r o b i a l s t r a i n s d e r i v e d from p r e - i n c u b a t e d s u b - s a m p l e s w a s a b o u t t w i c e t h a t of i s o l a t e s p i c k e d from S W A i n o c u l a t e d s o o n a f t e r c o l l e c t i o n of t h e s e a w a t e r s a m p l e s . F r o m b o t h s o u r c e s d i s p r o p o r t i o n a t e l y l a r g e n u m b e r s of c o l o u r e d i s o l a t e s w e r e lost.

D I S C U S S I O N

Z o B e l l (1946, p. 83) w r o t e : " S i n c e b a c t e r i o p h a g e is g e n e r a l l y f o u n d a s s o c i a t e d w i t h l a r g e n u m b e r s of r a p i d l y m u l t i p l y i n g b a c t e r i a , it is v e r y d o u b t f u l if t h e s p a r s e b a c t e r i a l p o p u l a t i o n c h a r a c t e r i s t i c of t h e o p e n o c e a n is c o n d u c i v e to t h e d e v e l o p m e n t or a c t i v i t y of b a c t e r i o p h a g e . " S i n c e then, t h e o c c u r r e n c e of b a c t e r i o p h a g e s in o c e a n w a t e r h a s b e e n d o c u m e n t e d o n l y t w i c e . H i d a k a (1977) r e p o r t e d t h e i s o l a t i o n of b a c t e r i o p h a g e s from s a m p l e s c o l l e c t e d in t h e s o u t h w e s t e r n p a r t of t h e Pacific O c e a n from d e p t h s b e t w e e n 1 a n d 200 m. A m o n g 576 b a c t e r i a l s t r a i n s 72 w e r e f o u n d to b e p h a g e - s e n s i t i v e , a n d the 72 b a c t e r i o p h a g e s i s o l a t e d w i t h t h e s e h o s t s c o u l d b e a s c r i b e d to 7 g r o u p s a c c o r d i n g to t h e i r h o s t r a n g e . J o h n s o n & S i e b u r t h (1978) o b s e r v e d b a c t e r i o p h a g e p a r t i c l e s b y t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y in p r e p a r a t i o n s o b t a i n e d b y c o n c e n t r a t i n g o p e n o c e a n w a t e r 2-5000 t i m e s .

C h a i n a (1965) i s o l a t e d 629 b a c t e r i a l s t r a i n s f r o m I n d i a n O c e a n w a t e r a n d s e d i m e n t ; 10 of t h e s e w e r e s u s c e p t i b l e to b a c t e r i o p h a g e s , b u t t h e i r a c t u a l s o u r c e w a s not g i v e n .

T h i s s c a r c i t y of i n f o r m a t i o n , at l e a s t in part, s e e m s to b e d u e to a g e n e r a l l a c k of i n t e r e s t in m a r i n e b a c t e r i o p h a g e s as d o c u m e n t e d b y t h e s m a l l o v e r a l l n u m b e r of p u b l i c a t i o n s d e a l i n g w i t h t h e s e v i r u s e s d u r i n g t h e l a s t 3 d e c a d e s . A d m i t t e d l y the r a r e n e s s of p h a g e p a r t i c l e s in t y p i c a l m a r i n e e n v i r o n m e n t s p o s e s t e c h n i c a l p r o b l e m s w h i c h s o m e t i m e s c a n b e o v e r c o m e o n l y b y c o n s i d e r a b l e effort a s d e m o n s t r a t e d b y the p r e s e n t p a p e r . H o w e v e r , t h e o c c u r r e n c e of m a r i n e b a c t e r i o p h a g e s i n d i c a t e s t h a t t h e s e v i r u s e s a r e a n i n t e g r a l p a r t of t h e i r e n v i r o n m e n t a n d , t h e r e f o r e , s h o u l d not b e n e g l e c t e d .

T h e m e t h o d r e p o r t e d in t h i s p a p e r is s p e c i f i c a l l y a d a p t e d to t h e n e e d s a n d l i m i t a - tions a b o a r d s h i p d u r i n g l o n g - r a n g e c r u i s e s as w e l l a s to t h e i n v e s t i g a t i o n of s e a w a t e r c o n t a i n i n g e x t r e m e l y l o w n u m b e r s of b a c t e r i a w h i c h c a n b e g r o w n o n p r e s e n t l y k n o w n m e d i a . O b v i o u s l y it c a n b e s i m p l i f i e d to s o m e d e g r e e . A c c o r d i n g to the o b s e r v a t i o n s c o m p i l e d in T a b l e 4, at l e a s t t h e p r e p a r a t i o n s p r o d u c e d from t h e p h a g e - e n r i c h m e n t c u l t u r e s a f t e r 1 d a y of i n c u b a t i o n c o u l d b e o m i t t e d s i n c e o n l y a b o u t 4 % of the host b a c t e r i a w o u l d h a v e b e e n m i s s e d p r o v i d e d t h a t from 3 s u b - s a m p l e s p r e p a r a t i o n s w e r e m a d e a f t e r 2 a n d 3 d a y s of i n c u b a t i o n . T h e u s e of s e v e r a l s u b - s a m p l e s is w i t h o u t d o u b t p r e f e r a b l e to t h a t of o n l y o n e s a m p l e .

B a c t e r i o p h a g e s from o c e a n w a t e r 13

d e n s i t i e s of b a c t e r i a l p o p u l a t i o n s o b s e r v e d w i t h s e a w a t e r s a m p l e s c o l l e c t e d e a s t a n d w e s t of t h e A z o r e s {Table 2), t h e first o n e s g e n e r a l l y c h a r a c t e r i z e d b y h i g h e r c o n c e n t r a - tions of CFU. As c o m p i l e d from t h e f i n d i n g s o b t a i n e d d u r i n g t h e p h a g e - d e t e c t i o n tests, b a c t e r i o p h a g e s w e r e f o u n d from S u b - s a m p l e s 2 a n d / o r 3 a l o n e w i t h o n l y 20 ( = 17.4 %) a m o n g 115 " e a s t e r n " h o s t b a c t e r i a b u t w i t h 36 (-- 37 %) a m o n g t h e 98 " w e s t e r n " hosts. This i n d i c a t e s t h a t t h e u s e of s e v e r a l s u b - s a m p l e s is m o r e a d v a n t a g e o u s t h e p o o r e r t h e b a c t e r i a l p o p u l a t i o n of a s e a w a t e r s a m p l e .

It is u n k n o w n , h o w e v e r , to w h a t e x t e n t p r e - i n c u b a t i o n of s u b - s a m p l e s w i t h o u t o r g a n i c n u t r i e n t s a d d e d is b e n e f i c i a l as c o m p a r e d w i t h t h e u s e of t h e s a m e n u m b e r of s u b - s a m p l e s all e n r i c h e d at z e r o t i m e . T h e e m p l o y m e n t of p r e - i n c u b a t i o n is b a s e d on t h e a s s u m p t i o n t h a t e x t r e m e l y s p a r s e b a c t e r i a l p o p u l a t i o n s m a y d e v e l o p w i t h tess d i s t u r - b a n c e of t h e i r s p e c i e s c o m p o s i t i o n , at l e a s t for s o m e t i m e , in s a m p l e s w i t h d e l a y e d n u t r i e n t a d d i t i o n t h a n in s a m p l e s e n r i c h e d at once, t h e r e b y r e d u c i n g t h e p r o b a b i l i t y of s o m e m e m b e r s of a p o p u l a t i o n b e i n g o u t - g r o w n b y others. A s a m a t t e r of fact, i n s e v e r a l c a s e s d i f f e r e n t b a c t e r i o p h a g e s w e r e f o u n d w i t h t h e s a m e b a c t e r i a l h o s t from t h e p r e - i n c u b a t e d s u b - s a m p l e s o n t h e o n e h a n d a n d t h e r e l a t e d S u b - s a m p l e 1 o n t h e other. F u r t h e r m o r e , w i t h 56 hosts b a c t e r i o p h a g e s w e r e f o u n d o n l y in p r e p a r a t i o n s m a d e from S u b - s a m p l e s 2 a n d / o r 3, a n d w i t h 27 a d d i t i o n a l b a c t e r i a l hosts, p h a g e s w e r e d e t e c t e d from m o r e p r e p a r a t i o n s p r o d u c e d from t h e s e s u b - s a m p l e s t h a n from S u b - s a m p l e 1. F i n a l l y , w i t h o n l y 34 out of 213 i s o l a t e d h o s t b a c t e r i a , b a c t e r i o p h a g e s w e r e d e t e c t e d o n l y from S u b - s a m p l e 1. N e v e r t h e l e s s , s i m i l a r r e s u l t s m i g h t h a v e b e e n o b t a i n e d if a l l 3 s u b - s a m p l e s h a d b e e n e n r i c h e d w i t h n u t r i e n t s at z e r o t i m e .

As c a n b e s e e n from T a b l e 3, c o n s i d e r a b l e d i f f e r e n c e s w e r e o b s e r v e d in r e g a r d to the efficiency of t h e m e t h o d s u s e d for p h a g e d e t e c t i o n . T h e y b e c o m e m o r e s t r i k i n g if t h e g e o g r a p h i c o r i g i n of t h e s e a w a t e r s a m p l e s is t a k e n into account. W i t h 21 ( = 18 %) of t h e 115 " e a s t e r n " h o s t s (see a b o v e ) v e r s u s 44 ( = 45 %) of t h e 98 " w e s t e r n " h o s t b a c t e r i a , b a c t e r i o p h a g e s c o u l d o n l y b e f o u n d b y t h e m o r e s e n s i t i v e d e t e c t i o n p r o c e d u r e e m p l o y - i n g t h e r i n g h o l d e r s . F r o m t h e s e o b s e r v a t i o n s it m u s t b e c o n c l u d e d t h a t w i t h t h e w e s t e r n s e a w a t e r s a m p l e s , w h i c h c o n t a i n e d p o o r e r b a c t e r i a l p o p u l a t i o n s t h a n t h e e a s t e r n o n e s (Table 2), b a c t e r i o p h a g e s w e r e e n r i c h e d on t h e a v e r a g e to a l e s s e r d e g r e e t h a n w i t h t h e e a s t e r n s a m p l e s . T h e r e f o r e , n o t o n l y t h e u s e of s e v e r a l s u b - s a m p l e s b u t a l s o t h e e m p l o y m e n t of m o r e s e n s i t i v e p h a g e - d e t e c t i o n p r o c e d u r e s is m o r e a d v a n t a g e o u s t h e p o o r e r t h e b a c t e r i a l c o n t e n t of t h e s e a w a t e r i n v e s t i g a t e d .

T h e p h a g e - d e t e c t i o n m e t h o d e m p l o y i n g t h e r i n g h o l d e r s w a s o r i g i n a l l y d e v e l o p e d for the u s e a b o a r d s h i p w h e r e u s u a l s p o t tests, e. g. t h e a p p l i c a t i o n of s e v e r a l d r o p s p e r p l a t e b y loops, w e r e f o u n d to b e u n r e l i a b l e b e c a u s e d r o p s w o u l d f l o w t o g e t h e r b e f o r e b e i n g a b s o r b e d b y t h e agar. S i n c e , h o w e v e r , t h e t e s t t a k e s m o r e t i m e t h a n w a s a v a i l a b l e d u r i n g the cruise, it w a s u s e d o n l y a f t e r r e t u r n . A l t h o u g h t i m e c o n s u m i n g , it is p r e f e r a b l e to o t h e r m e t h o d s of c o m p a r a b l e s e n s i t i v i t y b e c a u s e it r e d u c e s the d e m a n d for e q u i p m e n t (such as h u n d r e d s of t u b e s w h i c h w o u l d r e q u i r e c l e a n i n g ) , n e e d s o n l y s m a l l a m o u n t s of e x p e n s i v e m e d i a , a n d , i n t h e l o n g run, s a v e s t i m e .

14 K. M o e b u s

e q u i p m e n t s e v e r e l y h a m p e r e d b y a n a b n o r m a l l y h a r s h w i n t e r . L a s t b u t n o t l e a s t , t h e d e p e n d a b l e h e l p a n d c o m p a n i o n s h i p of t h e c r e w of RV " F r i e d r i c h H e i n c k e " a n d t h e s c i e n t i f i c staff d u r i n g t h e c r u i s e is c o r d i a l l y a c k n o w l e d g e d .

L I T E R A T U R E C I T E D

C h a i n a , P. N., 1965. S o m e r e c e n t s t u d i e s o n m a r i n e b a c t e r i o p h a g e s . - J . g e n . M i c r o b i o l . 41, Proc. 25. H i d a k a , T., 1971. I s o l a t i o n of m a r i n e b a c t e r i o p h a g e s f r o m s e a w a t e r . - Bull. J a p . Soc. s c i e n t . Fish.

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H i d a k a , T., 1977. D e t e c t i o n a n d i s o l a t i o n of m a r i n e b a c t e r i o p h a g e s y s t e m s i n t h e s o u t h w e s t e r n p a r t of t h e P a c i f i c O c e a n . - M e m . Fac. Fish., K a g o s h i m a U n i v . 26, 5 5 - 6 2 .

J o h n s o n , P. W. & S i e b u r t h , J. M c N . , 1978. M o r p h o l o g y of n o n - c u l t u r e d b a c t e r i o p l a n k t o n f r o m e s t u a r i n e , s h e l f a n d o p e n o c e a n w a t e r s . - A b s t r . a. M e e t . A m . Soc. M i c r o b i o l . N 95, 178. S p e n c e r , R., 1955. A m a r i n e b a c t e r i o p h a g e . - N a t u r e , L o n d . 175, 690.

S p e n c e r , R., 1960. I n d i g e n o u s m a r i n e b a c t e r i o p h a g e s . - J. Bact. 79, 614.