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A ‘capillary racetrack’ method for isolation of magnetotactic bacteria
FEMS Microbiology Ecology 45 (1987) 31-35 Pubhshed by Elsevier
31
FEC 00100
A 'capillary racetrack' method for isolation of magnetotactic bacteria R.S. W o l f e a, R . K . T h a u e r b a n d N. P f e n n i g c Department of Mt~robwlog~v, Unn,ersm' of Ilhnots, Urhana, IL U.S A., b MIkrobtologte / FB Btologw, Phzhpps Umt,er~ttiit, Marburg, F.R G., and ~ Fakultiit ffir Btologle, Um~'erstth't Konstanz, Konstanz, F R G
Received 30 October 1986 Accepted 12 November 1986
Key words: Magnetotactic bacterium; Magnetic field; Motility; (Isolation method)
1. S U M M A R Y A capillary tube was developed in which an inoculum of magnetotactic bacteria that contained only a few contaminants could be separated from crude sediment in a few minutes. Sterile fluid was placed on one side of a wetted cotton plug and sediment was placed on the other side. Magnetotactic bacteria migrated quickly through the cotton toward the south pole of a stirring-bar magnet placed at the closed end of the capillary. Protozoa and chemotactic bacteria were significantly delayed in passage through the cotton. 2. I N T R O D U C T I O N Although it has been over a decade since the first magnetotactic bacterium was discovered [1], to our knowledge the isolation of only one species in pure culture has been reported [2]. Sediments of eutrophic lakes and ponds may contain high numbers of a variety of these organisms [3-6] and laboratory enrichments with sediments may be readily established. In some cases the organisms are in such high concentrations that they may be Correspondence to: R.S. Wolfe, Dept. of Microbiology, University of Illinois, Urbana, IL 61801, U.S.A
collected from the wall of the container by means of a magnet [4,6]. The present investigation was begun in an attempt to develop a simple procedure for procuring an enriched inoculum of magnetotactic organisms from freshly collected sediment or laboratory enrichments, the strategy being to impose a magnetic field in such a way that these organisms would migrate out of the sediment into sterile medium where they could be collected and subjected to isolation by standard aseptic techniques. 3. M A T E R I A L S A N D M E T H O D S
3.1. Preparation of the captllary The drawn end of a Pasteur pipette was marked with an ampule file as shown in Fig. 1A, and broken off. The large end of the pipette was ringed by marking with an ampule file (Fig. 1A) and broken by touching the mark with the melted end of a glass rod, leaving a section from the middle of the pipette that was about 8 cm long. The small end of this section was closed by melting the glass in a flame. Over the pilot flame of a bunsen burner, the section was bent very slightly at the arrow (Fig. 1A, b) so that when placed on a flat surface it assumed the position shown in Fig. 1A, b, the reservoir end being at a slight angle
0168-6496/87/$03.50 ~ 1987 Federation of European Microbiological Socmtlcs
32
J_l
.~
B
~/
~
~ ~ / ~
SI,de Plashc Shde
C 22gouge, lOcm
Fig 1 Method of constructaon of the 'racetrack' For details, see section 3 1
a b o v e the surface. A small piece of m e d i c a l abs o r b e n t c o t t o n was p l a c e d in the reservoir end of the c a p i l l a r y a n d very loosely p a c k e d into the n a r r o w neck. The b r o k e n end of the c a p i l l a r y (Fig. 1A, a) was used for this purpose. T h e c o t t o n b a r r i e r was not c o m p r e s s e d at this stage so that a h y p o d e r m i c needle (see section 3.3) could easily p e n e t r a t e it. The c a p i l l a r y was p l a c e d in a stand a r d test t u b e that was closed with a c o t t o n p l u g or plastic c a p a n d autoclaved.
3.2. Preparation of the holder F r o m a 3 - m m - t h i c k piece of Plexiglas the size of a s t a n d a r d m i c r o s c o p e slide, the center section was cut out as shown in Fig. 1B, b. A glass slide was then glued to each side (Fig. 1B, a, b, c) with a glue used for h o u s e h o l d p o r c e l a i n repair.
3. 3. Aseptic filling of the capillary A syringe was filled with water and some sedim e n t from the t o p area of sediment. A sterile 0.2 /~m filter unit ( M i l l e x - G V , M i l l i p o r e Corp., Bedford, M A ) to which was a t t a c h e d a sterile 10-cm, 22 gauge stainless steel h y p o d e r m i c needle, was p l a c e d on the syringe. T h e syringe was held in a vertical p o s i t i o n as water was forced t h r o u g h the filter a n d into the needle so that the water was free of air b u b b l e s . The first 0.5 ml or m o r e from the needle was discarded. The sterile needle was then thrust u n d e r the c o t t o n plug to the closed e n d of the capillary, a n d the c a p i l l a r y a n d reservoir were filled with sterile water without any air b u b b l e s being t r a p p e d as the needle was w i t h d r a w n (Fig. 1C). If the c o t t o n plug was disl o d g e d as the needle was being removed, it was
33
repositioned using the needle or a sterile Pasteur pipette. (It was essential that the needle point should be straight and free of any burr.) At this time the sterile cotton was positioned more firmly in the capillary neck, and most of the water in the reservoir was removed with a Pasteur pipette.
3.4. Operation of the 'racetrack' An inoculum of sediment that contained magnetotactic bacteria was placed in the reservoir of the capillary tube (Fig. 2A, a), and the capillary was placed in the slide holder, which had been filled with distilled water. The slight bend in the capillary allowed it to make firm contact at points 1, 2, and 3 (Fig. 2A, a) forming a rigid structure. A stirring-bar magnet was positioned as shown (Fig. 2A, b) with the south pole next to the capillary holder. To preserve the relative position of the magnet and the holder, both were placed on a piece of clear plastic (3.5 × 12 × 0.35 cm) that was on a microscope stage. The mechanical stage could be removed and the plastic sheet moved by hand to position the capillary holder, or the plastic could be cut to fit in the mechanical stage. To ensure that the magnet remained in position and was not attracted to a metallic component of the microscope, it was held in place by a piece of adhesive tape.
4. RESULTS
4.1. The inoculum The capillary procedure was found to be effective only if cells were in an active physiological state. Sediment was assayed for the presence of active magnetotactic cells by microscopic examination of a drop on a slide as noted by Blakemore [1]. A new microscope slide was unsuitable for this purpose, since the drop spread into a thin layer. To keep the drop rounded and in one place, a microscope slide was treated by rubbing a finger over the surface so that natural oils coated the surface. The surface of the slide was then wiped with a tissue prior to use. Several samples of sediment could be examined at one time as each drop formed a rounded shape. When the south pole of a stirring bar magnet was positioned toward the drops on the microscope stage, magnetotactic cells accumulated at the edge of the drops. A concave slide was used in a similar manner by Blackmore [1]. The activity of the cells could be evaluated readily. In certain sediments it took up to 5 min for significant numbers of cells to appear.
4.2. Observation After a sample of fresh sediment had been
I
A
a
3
!
S,de View
I
2 cotton ] / ~inoculum
b
(.
©
~...~ ...;'..;'C".."..;'..:"..;".;'.,."."~'. ;'..2"..;"..%?'..% i~J~
Magnet
Top
J - ~ water
E~
/
f
water
~
,.f'~,
/~
.f
Fig 2 Operation of the 'racetrack'. For details, see section 3 4
V~ew
34 placed in the reservoir and the racetrack was ready to be used, it was essential that the surface of the capillary should be wiped carefully with tissue to remove fingerprint and debris before it was placed in the holder. Water in the holder prevented optical diffraction by the capillary surface and allowed clear observation of cells in the fluid. One of the best ways to observe magnetotactic cells as they migrated out of the cotton and down the capillary was found to be dark-field illumination by use of a stage phase 3 setting with 10 × or 16 × phase ocular. A dissecting microscope also was found to be useful for following larger magnetotactic cells. Creeping and gliding magnetotactic bacteria also navigated through the cotton barrier, although it took them much longer to traverse the obstacle course. At certain times initially active cells gradually slowed their motility as they proceeded down the capillary, exhibiting stress and ceasing motility. This behavior was traced to the solution placed in the capillary. Highly aerobic solutions quickly inactivated the magnetotactic cells. After testing a variety of buffers and reducing agents, the most reliable racetrack fluid was found to be water associated with the surface layer of sediment from which the inoculum was to be taken. The open end of a 2-cc syringe was used to disturb the surface of the sediment, and as it settled the syringe was filled with water and sediment from this area. The syringe was immediately attached to a sterile filter, air was displaced from the filter with water from the syringe, and about 0.5 ml was discarded through the needle. This solution was found to be stable in the syringe, and could be used over a period of 30 min, providing an effective capillary fluid in which magnetotactic cells migrated well. The filled capillary was examined carefully to ensure that no air bubbles were present in the capillary fluid. Highly motile magnetotactic cells from an active sediment source easily arrived at the end of the capillary in a few minutes. The time required depended on the size and compactness of the cotton plug as well as on the activity of the cells. Cells were found to move in a continuous stream that could last 10 min or more with many hundreds
of cells accumulating in the capillary tip. Location of the microbial stream of cells in the capillary fluid depended on the position of the magnet (the magnetic lines of force). Cells could be most easily followed along the top of the capillary.
4. 4. Hart~estmg of the moculum When a desired inoculum of cells had accumulated, the capillary was removed from the holder such that the tip remained oriented toward the south pole of the magnet. The capillary was marked lightly about 2 cm from the tip with a diamond point, ampule file or the sharp edge of a piece of sandpaper, and then wiped with alcohol. The capillary was broken and the contents were removed with a drawn, sterile Pasteur pipette as shown in Fig. 2B. The inoculum was then used in standard aseptic procedures.
5. DISCUSSION The capillary racetrack method has the advantage that a highly purified inoculum of magnetotactic cells in a very active physiological state may be obtained within a few minutes. Although the method of Moench and Konetzka [4] produces a concentration of magnetotactic ceils, in our hands there were significant contaminants in the inoculum and the cells exhibited considerable physiological stress, as they had been collected in the aerobic aqueous area of the enrichment vessel. Magnetotactic cells so far studied appear to be microaerophiles. Microbiological techniques for the enrichment and isolation of gradient-dependent organisms such as microaerophiles are limited. When the surface of a sterile, reduced semi-solid agar medium is exposed to air, a gradient is established between the aerobic and anaerobic portions of the medium. A pure culture of a microaerophile may readily establish a dense population on the gradient and command the access of oxygen to the reduced medium; the presence of contaminants that can do this have so far prevented the successful use of semi-solid media for the isolation of magnetotactic bacteria. The method described here provides an inoculum of highly active cells that may be free of contami-
35
nant. It is obvious that the sooner the inoculum is harvested from the capillary tip, the higher the probability is that it may be free of non-magnetic cells. Although the method works best with highly motile cells, magnetotactic cells with other types of motility (gliding, twitching) could also be enriched in the fluid of the capillary.
ACKNOWLEDGEMENTS This method was developed when R.S.W. was a guest in the laboratories of R.K Thauer and N. Pfennig, and was supported by grants from the Deutsche Forschungsgemeinschaft to these investigators. R.S.W. was the recipient of an Alexander Von Humboldt Award.
REFERENCES [1] Blakemore, R. (1975) Magnetotactic Bacteria. Science 190, 377-379. [2] Blakemore, R.P., Maratea, D. and Wolfe, R.S. (1979) Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium. J Bacteriol. 140, 720-729 [3] Blakemore, R. (1982) Magnetotactic Bacteria. Annu. Rev Mlcrobiol. 36, 217-238 [4] Moench, T T and Konetzka, W A. (1978) A novel method for isolation and study of a magnetotactlc bacterium. Arch Microbiol. 119, 203-212. [5] Blakemore, R.P. and Frankel, R.B. (1981) Magnetic navigation in bacteria. Sci. Am. 245, 58-65 [6] Spormann, A M and Wolfe, R.S. (1984) Chemotactic, magnetotactic and tactile behavior m a magnetic spirillum. FEMS Microblol. Lett. 22, 171- 177.
31
FEC 00100
A 'capillary racetrack' method for isolation of magnetotactic bacteria R.S. W o l f e a, R . K . T h a u e r b a n d N. P f e n n i g c Department of Mt~robwlog~v, Unn,ersm' of Ilhnots, Urhana, IL U.S A., b MIkrobtologte / FB Btologw, Phzhpps Umt,er~ttiit, Marburg, F.R G., and ~ Fakultiit ffir Btologle, Um~'erstth't Konstanz, Konstanz, F R G
Received 30 October 1986 Accepted 12 November 1986
Key words: Magnetotactic bacterium; Magnetic field; Motility; (Isolation method)
1. S U M M A R Y A capillary tube was developed in which an inoculum of magnetotactic bacteria that contained only a few contaminants could be separated from crude sediment in a few minutes. Sterile fluid was placed on one side of a wetted cotton plug and sediment was placed on the other side. Magnetotactic bacteria migrated quickly through the cotton toward the south pole of a stirring-bar magnet placed at the closed end of the capillary. Protozoa and chemotactic bacteria were significantly delayed in passage through the cotton. 2. I N T R O D U C T I O N Although it has been over a decade since the first magnetotactic bacterium was discovered [1], to our knowledge the isolation of only one species in pure culture has been reported [2]. Sediments of eutrophic lakes and ponds may contain high numbers of a variety of these organisms [3-6] and laboratory enrichments with sediments may be readily established. In some cases the organisms are in such high concentrations that they may be Correspondence to: R.S. Wolfe, Dept. of Microbiology, University of Illinois, Urbana, IL 61801, U.S.A
collected from the wall of the container by means of a magnet [4,6]. The present investigation was begun in an attempt to develop a simple procedure for procuring an enriched inoculum of magnetotactic organisms from freshly collected sediment or laboratory enrichments, the strategy being to impose a magnetic field in such a way that these organisms would migrate out of the sediment into sterile medium where they could be collected and subjected to isolation by standard aseptic techniques. 3. M A T E R I A L S A N D M E T H O D S
3.1. Preparation of the captllary The drawn end of a Pasteur pipette was marked with an ampule file as shown in Fig. 1A, and broken off. The large end of the pipette was ringed by marking with an ampule file (Fig. 1A) and broken by touching the mark with the melted end of a glass rod, leaving a section from the middle of the pipette that was about 8 cm long. The small end of this section was closed by melting the glass in a flame. Over the pilot flame of a bunsen burner, the section was bent very slightly at the arrow (Fig. 1A, b) so that when placed on a flat surface it assumed the position shown in Fig. 1A, b, the reservoir end being at a slight angle
0168-6496/87/$03.50 ~ 1987 Federation of European Microbiological Socmtlcs
32
J_l
.~
B
~/
~
~ ~ / ~
SI,de Plashc Shde
C 22gouge, lOcm
Fig 1 Method of constructaon of the 'racetrack' For details, see section 3 1
a b o v e the surface. A small piece of m e d i c a l abs o r b e n t c o t t o n was p l a c e d in the reservoir end of the c a p i l l a r y a n d very loosely p a c k e d into the n a r r o w neck. The b r o k e n end of the c a p i l l a r y (Fig. 1A, a) was used for this purpose. T h e c o t t o n b a r r i e r was not c o m p r e s s e d at this stage so that a h y p o d e r m i c needle (see section 3.3) could easily p e n e t r a t e it. The c a p i l l a r y was p l a c e d in a stand a r d test t u b e that was closed with a c o t t o n p l u g or plastic c a p a n d autoclaved.
3.2. Preparation of the holder F r o m a 3 - m m - t h i c k piece of Plexiglas the size of a s t a n d a r d m i c r o s c o p e slide, the center section was cut out as shown in Fig. 1B, b. A glass slide was then glued to each side (Fig. 1B, a, b, c) with a glue used for h o u s e h o l d p o r c e l a i n repair.
3. 3. Aseptic filling of the capillary A syringe was filled with water and some sedim e n t from the t o p area of sediment. A sterile 0.2 /~m filter unit ( M i l l e x - G V , M i l l i p o r e Corp., Bedford, M A ) to which was a t t a c h e d a sterile 10-cm, 22 gauge stainless steel h y p o d e r m i c needle, was p l a c e d on the syringe. T h e syringe was held in a vertical p o s i t i o n as water was forced t h r o u g h the filter a n d into the needle so that the water was free of air b u b b l e s . The first 0.5 ml or m o r e from the needle was discarded. The sterile needle was then thrust u n d e r the c o t t o n plug to the closed e n d of the capillary, a n d the c a p i l l a r y a n d reservoir were filled with sterile water without any air b u b b l e s being t r a p p e d as the needle was w i t h d r a w n (Fig. 1C). If the c o t t o n plug was disl o d g e d as the needle was being removed, it was
33
repositioned using the needle or a sterile Pasteur pipette. (It was essential that the needle point should be straight and free of any burr.) At this time the sterile cotton was positioned more firmly in the capillary neck, and most of the water in the reservoir was removed with a Pasteur pipette.
3.4. Operation of the 'racetrack' An inoculum of sediment that contained magnetotactic bacteria was placed in the reservoir of the capillary tube (Fig. 2A, a), and the capillary was placed in the slide holder, which had been filled with distilled water. The slight bend in the capillary allowed it to make firm contact at points 1, 2, and 3 (Fig. 2A, a) forming a rigid structure. A stirring-bar magnet was positioned as shown (Fig. 2A, b) with the south pole next to the capillary holder. To preserve the relative position of the magnet and the holder, both were placed on a piece of clear plastic (3.5 × 12 × 0.35 cm) that was on a microscope stage. The mechanical stage could be removed and the plastic sheet moved by hand to position the capillary holder, or the plastic could be cut to fit in the mechanical stage. To ensure that the magnet remained in position and was not attracted to a metallic component of the microscope, it was held in place by a piece of adhesive tape.
4. RESULTS
4.1. The inoculum The capillary procedure was found to be effective only if cells were in an active physiological state. Sediment was assayed for the presence of active magnetotactic cells by microscopic examination of a drop on a slide as noted by Blakemore [1]. A new microscope slide was unsuitable for this purpose, since the drop spread into a thin layer. To keep the drop rounded and in one place, a microscope slide was treated by rubbing a finger over the surface so that natural oils coated the surface. The surface of the slide was then wiped with a tissue prior to use. Several samples of sediment could be examined at one time as each drop formed a rounded shape. When the south pole of a stirring bar magnet was positioned toward the drops on the microscope stage, magnetotactic cells accumulated at the edge of the drops. A concave slide was used in a similar manner by Blackmore [1]. The activity of the cells could be evaluated readily. In certain sediments it took up to 5 min for significant numbers of cells to appear.
4.2. Observation After a sample of fresh sediment had been
I
A
a
3
!
S,de View
I
2 cotton ] / ~inoculum
b
(.
©
~...~ ...;'..;'C".."..;'..:"..;".;'.,."."~'. ;'..2"..;"..%?'..% i~J~
Magnet
Top
J - ~ water
E~
/
f
water
~
,.f'~,
/~
.f
Fig 2 Operation of the 'racetrack'. For details, see section 3 4
V~ew
34 placed in the reservoir and the racetrack was ready to be used, it was essential that the surface of the capillary should be wiped carefully with tissue to remove fingerprint and debris before it was placed in the holder. Water in the holder prevented optical diffraction by the capillary surface and allowed clear observation of cells in the fluid. One of the best ways to observe magnetotactic cells as they migrated out of the cotton and down the capillary was found to be dark-field illumination by use of a stage phase 3 setting with 10 × or 16 × phase ocular. A dissecting microscope also was found to be useful for following larger magnetotactic cells. Creeping and gliding magnetotactic bacteria also navigated through the cotton barrier, although it took them much longer to traverse the obstacle course. At certain times initially active cells gradually slowed their motility as they proceeded down the capillary, exhibiting stress and ceasing motility. This behavior was traced to the solution placed in the capillary. Highly aerobic solutions quickly inactivated the magnetotactic cells. After testing a variety of buffers and reducing agents, the most reliable racetrack fluid was found to be water associated with the surface layer of sediment from which the inoculum was to be taken. The open end of a 2-cc syringe was used to disturb the surface of the sediment, and as it settled the syringe was filled with water and sediment from this area. The syringe was immediately attached to a sterile filter, air was displaced from the filter with water from the syringe, and about 0.5 ml was discarded through the needle. This solution was found to be stable in the syringe, and could be used over a period of 30 min, providing an effective capillary fluid in which magnetotactic cells migrated well. The filled capillary was examined carefully to ensure that no air bubbles were present in the capillary fluid. Highly motile magnetotactic cells from an active sediment source easily arrived at the end of the capillary in a few minutes. The time required depended on the size and compactness of the cotton plug as well as on the activity of the cells. Cells were found to move in a continuous stream that could last 10 min or more with many hundreds
of cells accumulating in the capillary tip. Location of the microbial stream of cells in the capillary fluid depended on the position of the magnet (the magnetic lines of force). Cells could be most easily followed along the top of the capillary.
4. 4. Hart~estmg of the moculum When a desired inoculum of cells had accumulated, the capillary was removed from the holder such that the tip remained oriented toward the south pole of the magnet. The capillary was marked lightly about 2 cm from the tip with a diamond point, ampule file or the sharp edge of a piece of sandpaper, and then wiped with alcohol. The capillary was broken and the contents were removed with a drawn, sterile Pasteur pipette as shown in Fig. 2B. The inoculum was then used in standard aseptic procedures.
5. DISCUSSION The capillary racetrack method has the advantage that a highly purified inoculum of magnetotactic cells in a very active physiological state may be obtained within a few minutes. Although the method of Moench and Konetzka [4] produces a concentration of magnetotactic ceils, in our hands there were significant contaminants in the inoculum and the cells exhibited considerable physiological stress, as they had been collected in the aerobic aqueous area of the enrichment vessel. Magnetotactic cells so far studied appear to be microaerophiles. Microbiological techniques for the enrichment and isolation of gradient-dependent organisms such as microaerophiles are limited. When the surface of a sterile, reduced semi-solid agar medium is exposed to air, a gradient is established between the aerobic and anaerobic portions of the medium. A pure culture of a microaerophile may readily establish a dense population on the gradient and command the access of oxygen to the reduced medium; the presence of contaminants that can do this have so far prevented the successful use of semi-solid media for the isolation of magnetotactic bacteria. The method described here provides an inoculum of highly active cells that may be free of contami-
35
nant. It is obvious that the sooner the inoculum is harvested from the capillary tip, the higher the probability is that it may be free of non-magnetic cells. Although the method works best with highly motile cells, magnetotactic cells with other types of motility (gliding, twitching) could also be enriched in the fluid of the capillary.
ACKNOWLEDGEMENTS This method was developed when R.S.W. was a guest in the laboratories of R.K Thauer and N. Pfennig, and was supported by grants from the Deutsche Forschungsgemeinschaft to these investigators. R.S.W. was the recipient of an Alexander Von Humboldt Award.
REFERENCES [1] Blakemore, R. (1975) Magnetotactic Bacteria. Science 190, 377-379. [2] Blakemore, R.P., Maratea, D. and Wolfe, R.S. (1979) Isolation and pure culture of a freshwater magnetic spirillum in chemically defined medium. J Bacteriol. 140, 720-729 [3] Blakemore, R. (1982) Magnetotactic Bacteria. Annu. Rev Mlcrobiol. 36, 217-238 [4] Moench, T T and Konetzka, W A. (1978) A novel method for isolation and study of a magnetotactlc bacterium. Arch Microbiol. 119, 203-212. [5] Blakemore, R.P. and Frankel, R.B. (1981) Magnetic navigation in bacteria. Sci. Am. 245, 58-65 [6] Spormann, A M and Wolfe, R.S. (1984) Chemotactic, magnetotactic and tactile behavior m a magnetic spirillum. FEMS Microblol. Lett. 22, 171- 177.