Mierob Ecol (1986) 12:331-341

MICROBIAL ECOLOGY 9 Springer-Verlag New York Inc. 1986

Methods and Sources for the Enrichment and Isolation of Budding, Nonprosthecate Bacteria from Freshwater Peter Hirsch and Michael Miiller* Institut fiir Allgemeine Mikrobiologie, Universilfit Kiel (Biozentrum); Olshausenstr. 40/60, D-2300 KIEL, Federal Republic of Germany

Abstract. Methods are described for the observation, enrichment and isolation (from various freshwater samples) of bacteria of the genera Planctornyces and Pirella. Because immature buds were easily dislodged by shearing forces, slide culture techniques and direct microscopy of the budding process are recommended. An "auxanographic" technique to detect possible stimulation by soluble substrates was based on the diffusion of these substrates from peripherally placed crystals into the agar of a slide culture. Nearly every water sample investigated contained representatives of the genera Planctomyces, Pirella, and Blastobacter, as well as budding cocci. Six enrichment techniques were tried; some enrichment experiments lasted several months. Allowing samples without added substrate to stand for a long time or generally employing nutrient-poor media were most successful. The "petri dish method," taking advantage of attachment of many budding bacteria to glass surfaces, was especially useful for increasing the numbers of Planctomyces spp. Pure cultures obtained from freshwater samples were tentatively placed in the genera Planctomyces, Pirella, and Blastobacter. One strain appeared to represent a new genus of gram-positive, budding, and nonprosthecate bacteria.

Introduction Budding as a process of bacterial daughter cell formation was recognized by Henrici and Johnson [6], who observed a variety of cell forms (morphotypes) with short stalks on one cell pole and buds on the other. Some o f these bacteria Were named Blastocaulis sphaerica and others identified as Pasteuria ramosa. The former has been shown to be a later synonym of Planctomyces bekefii Gimesi 1924 [7], a bacterium that was initially thought to be a planktonic fungus. A "'Pasteuria ramosa'" had been described by Metchnikoff [18] as an exospore-forming parasite of cladocerans, and Henrici and Johnson [6] considered one of their bacteria to be identical to it. Staley [26] supported this assumption when he described a new isolate resembling these forms. Recent observations [23] clearly showed P. ramosa to be substantially different from * P r e s e n t address:

Landes-Hygiene-lnstitut Oldenburg; Am Datum 46, 2900 Oldenburg.

332

P. Hirseh and M. Miiller

the organisms seen previously [6, 26], but identical to Metchnikoff's original description [ 18]. T h e strain isolated b y Staley [26] was therefore r e n a m e d Pirella staleyi [22]. In recent years a considerable a m o u n t o f w o r k has been d o n e on gramnegative hyphal a n d budding bacteria [9, 19]. H o w e v e r , knowledge on budding, n o n p r o s t h e c a t e bacteria is still scarce. C o m p a r a t i v e l y few pure cultures have been obtained [1, 4, 26, 28, 29], but b u d d i n g stalked or n o n s t a l k e d gramnegative bacteria h a v e been frequently o b s e r v e d in natural s a m p l e s [3, 10, 12, 14, 23, 27], including freshwater, brackishwater, hypersaline lakes, groundwater, a n d bogwater. H o w e v e r , p e r h a p s m o r e often b u d d i n g o f these bacteria m a y h a v e been overlooked; size differences o f one o r g a n i s m m a y h a v e been interpreted as different organisms, b u d s m a y h a v e b e e n dislodged b y preparation m e t h o d s , or the m i c r o s c o p i c p r e p a r a t i o n techniques m a y n o t h a v e allowed the necessary resolution for detection o f smaller buds. Recently, two i n d e p e n d e n t o b s e r v a t i o n s h a v e created renewed interest in budding, nonprosthecate bacteria. First, in cataloging 16S r R N A base sequences o f new Planctomyces a n d Pirella isolates o b t a i n e d by H. Schlesner (Ph.D. thesis, Kiel 1982), S t a c k e b r a n d t et al. [24] o b s e r v e d e x t r e m e l y low phylogenetic relationships o f these with all other eubacteria tested so far. In fact, Planctomyces and Pirella a p p e a r e d to represent an ancient b r a n c h o f eubacteria. T h e second surprise was the o b s e r v a t i o n that Planctomyces and Pirella strains completely lacked peptidoglycan; instead they h a d proteinaceous cell envelopes resistant to 10% SDS [17]. This p a p e r describes observation, e n r i c h m e n t , a n d isolation techniques for aquatic, n o n p r o s t h e c a t e budding bacteria. Since m i c r o m a n i p u l a t i o n is tedious a n d requires special experience, as well as the instrument, we preferred easier techniques. E m p l o y i n g these, we succeeded in isolating several new bacteria of this group; t h e i r f o r m a l descriptions h a v e been published elsewhere [30, 3 I].

Methods

Sampling Water samples were taken from the neuston down to 10 m in spring, summer, and autumn. TheY were drawn with sterilized flasks, transported to the laboratory over ice, and stored at 6~ Samples from greater depths were obtained with a Van Dorn sampler.

Microscopic Observations Several techniques were used to see bacteria with buds still attached. In many cases shearing forces easily separated even incomplete buds from their mother cells, so wide-mouthed pipettes and gentle handling techniques had to be employed. The observation techniques (Table 1) were as follows: O-I: Coarse methods involved mechanical separation from other bacteria, such as scraping bacteria off their attachment substrata with sterile spatulas or glass rods. O-II: Stepwise centrifugation of 20 liter water samples in sterile plastic bottles (15,000 x g, 20 min, 4"C, RCIIB, Sorvall, Newton, Conn.) resulted in a concentrated sediment that could be viewed by phase contrast microscopy.

Enrichment of Budding Bacteria

333

o-III: Water samples were successively filtered through 3, 0.8, and 0.2 #m Millipore membrane filters and the filtered organisms were resuspended in sterile sample water. O-IV: Gentler techniques involved concentration by natural sedimentation. Samples were stored for 1-2 days at 4"C. The sediment collecting at the bottom was then easily withdrawn while still COld and could be investigated by light or electron microscopy. O-V: There are many ways by which attaching organisms can be studied. The classical way is the direct microscopy of previously exposed glass slides [6, 8]. We exposed slides for 3-4 days in SUmmer and 7-9 days in winter, depending on the degree of eutrophication. The slides had to be dried rapidly by quickly draining them on tissue and gently blowing over them. They were then heat-fixed and finally remoistened with sterile sample water or saline solution before examination. O-VI: The "'petri dish method" [ 12] is the gentle transfer of a cover slip (which has been previously exposed to water) to an agar surface. For that, a large n u m b e r of sterile cover slips was pressed slightly and vertically into a layer of 2% water agar in a 20 cm diameter glass petri dish. Some of the cover slips had a coating of water agar. A water sample was then poured into the dish so that the upper edges of the cover slips remained free for handling. After several days, they were removed daily for phase microscopy. When interesting organisms had multiplied sufficiently, the agar layer of undisturbed cover slip could be placed directly onto a solid medium or streaked out on this solid surface. O-VII: Other cover slips (from Method O-VI) without an agar layer, but with organisms that had attached to them, were placed on sterile slides which had been precoated with various media e.g., MSS, containing 0.05% (w/v) D(+) glucose, 0.05% (w/v) Bacto Peptone, or 0.1% (w/v) D(+) galaetose. These were incubated in moist chambers and then studied with the phase microscope. O-VIII: A smiliar approach was used in a modified "auxanographic" technique [2]. We took Uncoated, but exposed cover slips from a petri dish experiment, rinsed these with physiological saline solution, and placed them in the center of an agar plate flat onto the surface; the media employed (HJM or 337, see below) lacked added carbon sources. Small crystals or droplets of Potential C-sources (e.g., acetamide, D(+) ribose, D(+) glucose, sodium formate) were then placed onto the medium next to the edge of the cover slips. After 4-9 days of incubation at 20"C, the COver slips were viewed in position using the phase contrast microscope. The objective was to detect rnicrocolony formation of any interesting organism in the vicinity of a given C-source. Multiplication near the carbon source was assumed to have been stimulated by its diffusion into the area underneath the cover slip. Control experiments, with no carbon sources, were done. O-IX: Water surface (neuston) organisms were collected with a large platinum loop [8]. O-X: Neuston was collected by touching the surface with clean glass slides or with a piece of household plastic film. The exposed side of the film was placed onto 2% wateragar-coated glass slides for optimal microscopy (N. Pfennig, personal communication).

Microscopy We used a Zeiss Photomicroscope II with Neofluar phase contrast optics and Optavar for exact Phase adjustment. This was absolutely necessary to find the often very thin stalklets of some of the budding forms.

Culture Media All chemicals used were of analytical grade (Merck, Darmstadt). Solid media contained 1.8% (w/ v) Bacto Agar (Difeo). The pH was adjusted after autoclaving. The media compositions were as follows: " P Y G V " medium (peptone-yeast extract-glucose-vitamin agar) and " P Y G " were prepared according to Staley [25]. Medium " 3 3 7 " has been described previously [11]. Medium " H J M " was a modification of one used by Henrici and Johnson [6] and contained (g l-t): KH2PO,.3H20 (0.125); MgSO4' 7H20 (0.125); Bacto Peptone (0.5); tap water, added to make 1,000 ml. Medium "MSS" contained (NH4)2SO4 (0.25); MgSO4.7H20 (0.25); KH2PO, (0.25); vitamin solution no. 6

334 Table 1. Observation technique 0-I 0-II

P. H i r s c h a n d M. Miiller O b s e r v a t i o n t e c h n i q u e s a n d presence o f b u d d i n g bacteria in freshwater habitats

Sampling

B u d d i n g bacteria observed

Frequency

Site

D e p t h (m)

Month

Forest P o n d , Michigan Lake Plussee, Holstein, G e r m a n y

0.3

5, 6

Planctomyces sp."

(+)

4

Blastobacter sp., b u d -

(+)

0-III

Lake Plussee

0-IV

W i n t e r g r e e n Lake, Mich.

0-IV

Cassidy Lake, Michigan

0-V

0--0.2

ding cocci 0-1.0 0.2-4.5

9

none

0

7, 8

P. bekefii

+++

P. guttaeformis, Pirella sp.

++

b u d d i n g cocci

+ + +

P. bekefii Pirella sp. Planctomyces sp." Hyphomicrobium sp. Blastobacter sp. Planctomyces s p )

++

0.5-1.5

7

Forest P o n d , Michigan

0-1.0

5-11

0-V

Lake Lansing, Michigan

0-1.0

3

0-V

Lake H~Sftsee, Holstein c

0--0.2

9

0-V

Lake Plussee c

neuston

9

0-VI 0-VI

Lake Plussee a Lake H~ftsee a

0-0.2 0-0.3

8 9

0-VI

Village P o n d , R 6 b s doff, Holstein, Germanya

0--0.3

0-VI

G r o u n d w a t e r , Segeberg Forest, Holstein

10

0-VI

Forest Pond, Michigan

+ 0-+ + + 0--+ + + 0--(+) +

+ ++

1

P. guttaeformis P. bekefii Planctomyces sp. a Hyphomicrobium sp. Planctomyces sp." Planctomyces sp. a Pirella sp. Planctomyces sp. ~

7

Planctornyces spp."

+ ++

0.4

7

Planctomyces sp."

+++

Planctomyces sp. a Planctomyces sp." Planctomyces s p ) Blastobacter sp. Planctomyces s p ) Planctomyces sp. a Hyphomicrobium sp.

0-VII

Kleiner Pl6ner See

0-0.2

9

0-VIII

Lake Plussee

0-0.5

8

0-IX

Lake H~Sftsee

neuston

10

0-IX

Lake H6ftsee

neuston

9

0-X

Forest Pond, Michigan

0-0.3

8

+ ++ ++ + +++ + ++ + ++ +

++ ++ + + + ++ + ++ +

M o r p h o t y p e III [23] b M o r p h o t y p e II [23] S a m p l e s stored for 3 weeks before slides were h u n g into the water d Samples stored for 1-4 m o n t h s before petri dish m e t h o d was started F r e q u e n c y estimations: 0 - - a b s e n t ; ( + ) - - r a r e l y found, b u t m o r e t h a n 5 individuals; + -- occasionally present; + + - - f r e q u e n t ; + + + - - v e r y frequent a n d d o m i n a t i n g the p o p u l a t i o n

Enrichment of Budding Bacteria

335

[27], 10 ml; carbon source (glucose, galactose, or peptone) (0.5-1.0); distilled water added to make 1,000 ml.

Enrichment Techniques Most enrichment experiments were carried out for several weeks or even months (Table 2). The Useof dilute media or lack of added carbon source often resulted in slow growth of budding bacteria, but this was preferred since previous experiments had shown that otherwise these were outnumbered by faster growing forms. The following techniques were employed: E-l: 100 ml samples were stored in sterile Erlenmeyer flasks (20"C, light or dark, aerobically) Without any addition. They were frequently examined for growth of interesting forms. E-2:20 liter samples were concentrated aseptically to a volume of 50 ml by continuous eentrifugation (4"12, RCIIB) and incubated at 20* (light or dark). Other samples treated the same way Were resuspended in 500 ml of sterile-filtered sample water before incubation. E-3:100 ml water samples were put in sterile 250 ml beakers, and sterile complex nutrients were added to a final concentration of 0.005-0.01% [25]. The beakers were covered with aluminum foil and incubated at 20"C and 1,300-20,000 lux. E-4: One milliliterportions of fresh or stored samples were inoculated directly into a large variety of media; incubation was at 200C or 30~ E-5: Sterile glass slides (method O-V) were hung into fresh or stored water samples and incubated in the light at 20"C. When budding bacteria were observed, the growth on the slides was scraped off with a sterile scalpel, streaked onto PYG medium, and incubated at 20"C. E-6: The petri dish technique described above also represents an enrichment technique [12].

Isolation Procedures Once enrichment cultures showed large numbers of budding bacteria (Fig. 1), subsamples were Spread on various oligotrophic media (PYGV, PYG, HJM, or 337 with 3.38 g/liter methylamine. HC1). Colonies appearing after 4-6 weeks were touched with sterile toothpicks and transferred to a master plate of the same composition as the enrichment medium and then to a labeled glass slide. If microscopic observation of the slide revealed an interesting form, the inoculum on the master plate was labeled, cultivated, and purified by subsequently streaking it onto solid media.

Results

Observation Techniques B u d d i n g a n d n o n p r o s t h e c a t e b a c t e r i a were f o u n d i n m a n y o f the s a m p l e s i n vestigated. M o s t o f t h e m r e s e m b l e d m e m b e r s o f t h e g e n u s Planctomyces ( m o r p h o t y p e s II, III, a n d V; [23]), Pirella [22], a n d Blastobacter ( r o d - s h a p e d b a c teria; [15]). T h e r e were also b u d d i n g cocci w i t h o u t stalks a n d p e a r - s h a p e d rosette f o r m e r s . A c o m p a r i s o n o f t h e m e t h o d s e m p l o y e d is s h o w n i n T a b l e 1. I m m e d i a t e a n d d i r e c t m i c r o s c o p y o f n a t u r a l w a t e r s a m p l e s rarely r e v e a l e d b u d d i n g f o r m s ; e v i d e n t l y these were v e r y u n c o m m o n i n the free state, a n d p e r h a p s t h e y were generally rare. S c r a p i n g off surface g r o w t h (O-I) likewise r a r e l y o b t a i n e d b u d d i n g f o r m s . C o n c e n t r a t i o n b y c e n t r i f u g a t i o n (O-II) or stepwise f i l t r a t i o n (OIII) c o n c e n t r a t e d o t h e r b a c t e r i a a n d w a s u n s u c c e s s f u l for i s o l a t i n g b u d d i n g

336 Table 2.

P. Hirsch and M. Miiller Enrichment techniques for aerobic budding bacteria from freshwater habitats

Enrichment technique a

Site

Light

Weeks

E-1

Lake H~Sftsee

Neuston

9

rtb

+

8

E- 1

Village pond, RSbsdorf (Holstein) Kleiner PlSner See Groundwater, Segeberg Forest, Holstein Lake Plussee

0.2

9

rt

-

4

Planctomyces sp. c stalked rods Stalked rods

0.2

9

rt

-

4

Stalked rods

10.0

7

9

-

6

Planctomyces sp. c

0.2

4

rt

+

9

Kleiner P16ner See Lake Lansing, Mich.

0.2

9

rt

+

4

0.5

12

23

-

7

E-4 E-5

Lake HSftsee Lake H/Sftsee, Lake Plussee

0.2 0.2

10 9

rt rt

1 4

E-5

Bog pond, Kaltenhofe, Holstein Lake Plussee

0.2

5

rt

4

0.2

8

rt

16

Groundwater, Segeberg Forest

10.0

7

9

8

Budding cocci, Blastobaeter sp. Planctomyces sp. a P. bekefii Planctomyees sp. c Pirella sp. Hyphomicrobium sp. Blastobacter sp. P. bekefiL P. guttaeformis, Planctomyces sp., ~ Hyphomicrobium sp. Budding, motile rods (gram-positive) Planetornyces sp. d Pirella sp. Planctomyces sp. d

E- l E- 1

E-2 E-3 E-3 (E- 1)

E-6 E-6

Incubation Temp. Depth (m) Month (~

Budding bacteria obtained in enrichments

a For details see Materials and Methods b Room temperature, 18-23~ Morphotype II [23] a Morphotype III [23] bacteria, since most budding forms lived in the attached state. Concentration by cold, natural sedimentation (O-IV) was more successful with anaerobic lakewater samples than with other sample types. By far the greatest diversity o f b u d d i n g f o r m s w a s o b s e r v e d w i t h t h e H e n r i c i a n d J o h n s o n t e c h n i q u e ([6]; m e t h o d O - V ) , a n d w i t h its v a r i o u s m o d i f i c a t i o n s , s u c h as c o a t i n g s w i t h v a r i o u s a g a r m e d i a p r i o r t o u s e [8]. E s p e c i a l l y r i c h c r o p s o f b u d d i n g f o r m s w e r e o b t a i n e d w i t h t h e p e t r i d i s h m e t h o d ([ 12]; O - V I ) . W i t h t h i s t e c h n i q u e , b u d d i n g b a c t e r i a w e r e f o u n d e v e n i n g r o u n d w a t e r s a m p l e s [13], w h e r e , i n s o m e e a s e s , 400/0 o f

Enrichmentof Budding Bacteria

337

microorganisms were budding forms. By incubating exposed cover slips from Petri dish experiments, we could follow the budding process where it could not be seen otherwise, because buds were easily dislocated from their mother cells. The auxanographic technique (O-VIII) likewise revealed the formation of buds and gave indications of the types of carbon sources used by organisms that Could not be cultivated otherwise. Acetamide and D ( - ) ribose, in particular, yielded budding of forms similar to Planctomyces and Pirella spp. In some habitats the water surface pellicle was especially rich in budding bacteria, which could be detected by methods O-IX and O-X.

Enrichment Techniques We employed six enrichment techniques, with variable results (Table 2). Most SUccessful were methods based on the use of nutrient-poor media or on the incubation of unamended samples for several months (Fig. l). Eventually, the budding, attaching bacteria were as numerous or even more frequent than rods, Cocci, or vibrios. Water samples that were incubated without additions (method E-l) initially showed blooms of vibrios or rods. Only after 4-8 weeks were budding forms (Planctomyces, Blastobacter) more numerous (Fig. 1). After several months, Usually Hyphomicrobium spp. were the predominating budding bacteria. Some of the budding forms appeared in illuminated enrichments only, although photosynthetic pigments were not present. A surface sample from Lake Hrftsee Stored for 3 weeks and then spread on PYGV medium yielded a pure culture of a Blastobacter (strain IFAM 1004). Incubating samples that were concentrated by centrifugation (method E-2) did not allow budding bacteria to develop any recognizable bloom. When such a concentrate was diluted 1:10 with filtered sample water and incubated at 20~ budding bacteria, including cocci and rods, appeared in large numbers after 5 weeks. Subculture on PYGV medium yielded a pure culture of another Blastobacter sp. (strain IFAM 1005). The addition of 0.01% Bacto Peptone to a sample from Lake Kleiner Plrner See and incubation for 2 weeks (20~ light) resulted in a bloom of Planctomyces bekefii (method E-3). Although this mass development persisted for another Week, we were not able to culture P. bekefii on solid media. Method E-3 also produced excellent growth of Caulobacter spp. which often formed large rosettes. Furthermore, with this method we succeeded in obtaining dense enrichments of a pear-shaped Planctomyces sp. (Figs. l a and b), a stalked bacterium first mentioned by Henrici and Johnson [6] under the name of "Blastocaulis sphaerica." Preliminary electron microscopic results indicated that this organism resembled morphotype II organisms described by Schmidt and Starr [23]. The pear-shaped Planctomyces did not develop further on agar media. After 3-4 weeks, method E-4 (inoculation of media with 1 ml of sample), yielded many budding bacteria in the water surface pellicle. When a 2-weekold surface sample from Lake Hrftsee was inoculated into medium HJM and incubated at room temperature, a Blastobacter population developed. Streaking

338

P. Hirsch and M. Mfiller

Fig. 1. Enrichments of budding, nonprosthecate bacteria from a 0-15 cm, December sample from Lake Lansing (Michigan). All cultures were incubated in the dark and at 23~ Phase contrast light micrographs; magnification 1,285 • (A) Pear-shaped Planctornyces sp. after 9 days incubation. No additions to the sample; technique E-I. The stalks are excretions and carry a terminal boldfast. (B) Pear-shaped Planctomyces sp. after 5 days incubation (technique E-l). (C) Pirella sp. after 49 days of culture; technique E-I. The budding Pirella spp. lack excreted stalks. (D) Aggregates or rosettes ofPirella sp. after 49 days of incubation. No additions to the sample (technique E-l). (E) Pear-shaped Planctomyces sp. after 9 days of culture. Sample enriched with 0.005% of sterile Bacto Peptone (technique E-3). The bars represent 10 gm.

ou~ on the same medium resulted in a further pure culture (Blastobacter sp., strain IFAM 1003). The incubation of glass slides in stored water samples without amendments (method E-5) was very useful. We stored H6ftsee surface water (collected in September) for 3 weeks at 20~ Then glass slides were suspended in this sample for 7 days and examined. A bloom of Planctomyces bekefii developed; there were also numerous bottle-shaped, spined organisms resembling "Planctomyces guttaeformis'" Hortobagyi [5, 16]; or morphotype V of Schmidt and Starr [23]. Using the same technique and a sample from Lake Plussee (Schleswig-Holstein, Germany), we obtained masses of short-stalked, pointed rods (as had been described by Henrici and Johnson, as well as Hyphornicrobiurn spp. When we sampled a small bog pond in the Kaltenhofer Moor (near Kiel; pH 5.0; June)

Enrichment of Budding Bacteria

339

and stored it for 6 months, n u m e r o u s budding bacteria grew. Subcultures on PYG m e d i u m at 20 ~ yielded a pure culture o f a motile, budding, gram-positive bacterium which apparently represented a new genus (IFAM 1006). The petri dish m e t h o d was especially valuable for the e n r i c h m e n t o f Planctornyces and Pirella ( m e t h o d E-6). We used some o f the c o v e r slips for the auxanographic observation technique (O-VIII). W h e n a petri dish was inoculated with a 4 - m o n t h - o l d water sample from Lake Plussee, we observed a Planctomyces b l o o m after only 24 hours incubation in the dark. Most o f the budding bacteria appeared to attach near the b o t t o m agar layer o f the petri dish. The agar layer o f exposed c o v e r slips was then transferred right side up to m e d i u m P Y G agar and incubated in the dark for 4 weeks. F r o m reddish colonies that developed, we isolated two strains o f Planctomyces spp. (IFAM 1007 and 1008). With the same technique we obtained a Pirella strain from a Lake H6ftsee surface water sample. Even with groundwater as the inoculum, budding bacteria could be seen and isolated [ 13].

Isolations With the use o f oligotrophic media, especially P Y G V , and employing the toothpick m e t h o d for isolating colonies, we were able to obtain the pure cultures of three new Blastobacter spp. (IFAM 1003-I005), a gram-positive budding rod (IFAM 1006), Planctomyces sp (IFAM 1008), and a Pirella sp., which was later lost again. This technique p r o v e d to be quite valuable since in a short time a very large n u m b e r o f colonies could be screened for morphological Specialties. The new organisms m e n t i o n e d here have been described and n a m e d elsewhere [30, 31].

Discussion Budding, nonprosthecate bacteria were found in nearly every freshwater sample. T h e y seemed to be more n u m e r o u s near the water surface or in the attached COmmunity. The question arises then, why do so m a n y budding forms produce holdfasts and attach or form rosettes? One could speculate that the reverse is true, i.e., that it could have been the attached state (preferred in an oligotrophic environment) that resulted in greater cell polarity and in local cell wall growth, distal from the point o f a t t a c h m e n t [20]. This, o f course, m a y also have resulted in the need for reproduction through the f o r m a t i o n o f motile swarmer cells. The lakes and ponds we successfully investigated varied widely with respect to pH, eutrophication, or depth. T h e y were located in G e r m a n y (Holstein) and the USA (Michigan). Thus worldwide distribution o f these organisms in m o d erate climates can be assumed. Similar observations have been reported by PIirsch and R h e i n h e i m e r [14]. While the time o f sampling appeared to be unimportant in the case o f Planctomyces and Pirella spp., we found Blastobacter spp. mainly during spring and autumn. Staley et al. [27] found most budding bacteria during the summer. F o r m e r studies have often failed to recognize the ubiquity o f budding bac-

340

P. Hirsch and M. Miiller

teria. This may be explained by the observation that with most budding bacteria the buds are easily dislodged from their mother cells, unless sampling and observation methods are gentle. It is, therefore, advisable to observe bud formation using microculture. In enrichments set up with rich media, the oligotrophic, budding forms appeared to multiply visibly only after several weeks or even months, perhaps after most organic nutrients were used up. This could be due to slow growth and inability to compete with more versatile copiotrophic bacteria. One could also speculate that special carbon and/or energy substrates that were needed were supplied by the faster-growing other organisms. But our pure cultures grew well with a variety o f c o m m o n carbon sources, which does not support this assumption [30, 31]. After viable cell counting with the M P N method, Staley et al. [27] came to the conclusion that Planctomyces-Pasteuria (=Pirella) group organisms were most numerous in eutrophic habitats. The removal of competitors in the dilution procedure may explain his observations. The budding bacteria we obtained did not display unusual colony forms and only a few were pigmented so they could not be distinguished from colonies of nonbudding forms. For this reason we had to employ the toothpick method to scan as large a number of colonies on the isolation plates as possible. However, with some patience the nonprosthecate budding bacteria could be found easily among the many others. Acknowledgments. Parts of this work were supported by a grant to P. Hirsch from the Deutsche Forschungsgemeinschaft. We gratefully acknowledge the technical assistance of B/irbel Hoffmann. Other parts of this work were taken from the Ph.D. Thesis of M. Mailer (Univ. Kiel, Germany, 1977).

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Methods and sources for the enrichment and isolation of budding, nonprosthecate bacteria from freshwater.

Methods are described for the observation, enrichment and isolation (from various freshwater samples) of bacteria of the generaPlanctomyces andPirella...
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