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A convenient method for maintaining Chloroflexus for long time periods as slow growing liquid cultures
ELSEVIER
Journal ofMicrobiological Methods
Journal of Microbiological Methods 27 ( 1996) 15I- 155
A convenient method for maintaining ChZoroJexus for long time periods as slow growing liquid cultures Khursheed
A. Malik*
DSMZ-Deutsche Sammlung van Mikroorganismen und Zellkulturen GtnbH, Mascheroder Weg lb, 38214 Braunschweig. Germany
Received 12 August 1996; accepted 19 August 1996
Abstract A simple procedure is described to maintain Chlor-&xus cultures for relatively long times without frequent subculturing. All tested strains of CJzlorojexus auruntiucus proved viable after more than 18 months of storage in dim light (100-200 lx)
at 37-40°C as slow growing standing liquid cultures. During storage, no shaking, regular subculturing or transfer to fresh medium was required. To increase the span of generation time, cell survival and stability, various growth components and adsorption materials were screened. The anaerobic conditions, presence of sulfide and regular feeding with yeast extract favored prolonged viability during maintenance as liquid cultures. The presence of activated charcoal (0.05% w/v) in the media resulted in slowing down the growth rate and due to the absorption of harmful metabolites and waste products, the cells remained more rstable and healthy during such maintenance. The cultures of Chloroflexus aggregans also survived several months under such conditions. Due to the use of moderate conditions, regrowth in fresh medium resulted within relatively short times with full pigmentation, indicating that the maintenance and cultivation conditions were optimal for photoautotrophic growth. The method is more suitable, effective and convenient than cryopreservation or lyophilization as the maintained cell suspensions can provide a continuous source of inocula, from the same batch of cell suspension, for routine work. Keywords:
ChZoroJlexm;
Slow growing;
Standing liquid cultures; Long-term
1. Introduction Difficulties in the cultivation and laboratory maintenance of phcltosynthetic flexibacteria Chloroflexus are well known [ 1,2]. Liquid pure cultures of Chlorojkxus are dlifficult to maintain at normal growth temperature, and light intensity. Although media and methods for their culturing have improved greatly over the past few years, their cultivation and maintenance still causes problems [3-61. Chloroplexus aurantiacus, the first genus and species of the *Fax: +49 531 2616418.
Adsorption
materials
family Chloroflexaceae is generally maintained as living culture with a bi-weekly subculturing interval involving a frequent shaking. To avoid frequent transferring, the use of a co-culture (by mixing the culture with a known pure strain of cyanobacterium) has also been reported [ 1,7]. However, to retrieve a pure culture, a cumbersome reisolation is required every time. Frequent regular subculturing of stocks has many disadvantages and by increasing the transfer interval, costly and labour intensive frequent serial subculturing can be avoided. The effective maintenance of Chlorojkus is thus desirable so that the maintained
0167.7012/96/$15,00 Copyright 0 1996 Elsevier Science B.V. All rights reserved PZZ SO167-7012(96)00942-6
maintenance;
K.A. Mulik I Jourtml oj Microbiologicul
152
cell suspensions are immediately available as a continuous source of inoculum. Some preliminary results showing the comparative effect of various maintenance conditions and several growth supplements on long-term maintenance of such liquid cultures have already been reported [3,4]. This study presents data on the successful maintenance of ChloroJexus aurantiacus and Chloroflexus aggregans for long time periods as slow growing living cultures without frequent subculturing.
2. Material
and methods
2. I. Microorganisms Four strains of Chlorojlexus aurantiacus (DSM 635, 636, 637 and 638) and two strains of Chloro@ems aggregans (DSM 9485 and 9486) were used throughout this work. All bacteria were from the German Collection of Microorganisms and Cell Cultures (DSMZ). 2.2. Preparation
of cultures
For maintenance, the cultures were grown under semiaerobic conditions (in lOO-ml flat glass bottles with screw-caps) in the conventional Medium 1 (described by Schmidt [g]) and under anaerobic conditions (in lOO-ml flat glass bottles with screw caps with holes for the injection of the samples and butyl rubber septa) in a modified Medium 2 (described by the author in detail [9], and listed in Table 1). For Chlorojlexus aggregans, 1.0 ml/l, sterile vitamin solution CA [lo] was added to Medium 1 and 2. The cultures were incubated at 50°C at a light intensity of 300-500 lx. 2.3. Preparation and additives
of growth components,
adsorbents
Solutions of yeast extract (10% w/v kept under N?) and 10.0% (w/v) neutral activated charcoal (from Caelo, 4010 Hilden, FRG or any other of medicinal grade) were prepared in distilled water, autoclaved at 115°C for 13 min and stored at 4°C. Neutralized sodium sulfide solution (1.5% w/v in H,O) was prepared in a closed screw-capped bottle
Methods -77 (1996) 151-155
Table I Modified Growth Medium 2 for Chloroj7aus Yeast extract 1.0 g 1.0 g Glycyl-glycine NazHP0,X2H20 0.1 g 0.1 g MgSO,X7H,O 0.1 g KNO, 0.5 g NaNO i 0.1 g NaCl 0.05 g CaClzX2Hz0 5.0 ml Ferric citrate solution (0.1 g in 100 ml HZO) I .O ml Trace element solution SL-6 1050.0 ml Distilled water The pH of this medium was adjusted to 8.2. The medium was boiled under a stream of N2 for a few minutes and 90 ml medium was distributed into 100.ml screw-capped bottles with screw caps having holes and butyl tubber septa. Each bottle was bubbled with N1 and closed immediately with a rubber septum and screw tightened. The bottles were autoclaved at 121°C for 15 min. After autoclaving. 1.0 ml of neutralized sulfide solution (0.015% end concentration [3]) was injected into each bottle. For Chforojlexus nggregam, 1.O ml/l, sterile vitamin solution CA [IO] was added. This medium can be stored for several months under anaerobic conditions. Trace element solution SL-6: ZnSO,X7H,O MnCI,X4HZ0 HISO, CoCl,x6H,O CuCI,X2H,O NiC12X6H,0 Na,MoO,X2H,O Distilled water
0.1 g 0.03 g 0.3 g 0.2 g 0.01 g 0.02 g 0.03 g 1000 ml
with a butyl rubber septum and pH was adjusted to about 7.3 by adding 2 M H,SO, drop-wise with a syringe without opening the bottle [3]. 2.4. Storage of liquid cultures For comparison, stock cultures were grown to the logarithmic growth phase in different media under three different conditions and were kept for storage under parallel conditions. A: Control, with no additives. Kept under semiaerobic conditions after growing in Medium 1 [S]. B: No additives. Kept under anaerobic conditions in the presence of sulfide after growing in modified Medium 2 [9]. C: Supplemented with 0.05% activated charcoal after good growth appeared in modified Medium 2. The addition of activated charcoal was avoided at the beginning as it slows down the growth. Cultures were kept
K.A. Malik I Journal of Microbiological
under anaerobic conditions in the presence of sulfide and activated charcoal. All the cultures were maintained under parallel conditions for more than 20 months at 37-40°C in dim light (loo-200 lx). The samples were regularly supplemented every 2 weeks (for the first 3 months) and later at monthly intervals (for 12-20 months) with yeast extract (0.1% end concentration using a l-ml syringe flushed with nitrogen gas). For viability assay from the maintained culture, 1.0 ml was withdrawn (using a sterile gas-tight 2-5 ml syringe) at regular intervals and serial decimal dilutions were prepared. After showing non-viability, the culture A (Table 2) was not supplemented further. For viability assay from the maintained cultures, 1.0 ml was withdrawn at regular intervals and serial decimal dilutions were prepared. 2.5. Recultivation
ofcultures and sterility test
For recultivation, about 1 ml of the stored cell suspension was transferred to 14 ml of fresh medium (in Hungate tube). The standing cultures were replenished with equal amounts of fresh medium. For sterility tests, cultures were streaked on normal agar medium plates and on a universal heterotrophic Table 2 Survival of Chlorof7exu.r liquid cultures
during long term maintenance
Media and conditions”
Strain
Methods 27 (1996) 151-155
153
medium H,P [ 111. The cultures were grown at 49°C under light (500-1000 lx).
2.6. Viability assay The viability assay was done first after every 3 weeks, later every 6 weeks and finally after every 6 months of storage. For precise determination of viable cell counts during storage, DSM 635, DSM 636 and DSM 9485 were selected. For the estimation of viable cell counts, 0.5 ml of inocula was transferred from the standing cultures (for reproducible results, a through mixing of the dilution tubes was necessary to eliminate clumps) and injected into 4.5 ml of prereduced medium in screw-cap tubes (Hungate tubes with septa, Bellco Glass Inc., 204716125) and 6-8 serial decimal dilutions were prepared using oxygen free syringes with incubation under appropriate conditions. The number of viable cells was determined using the most probable number method (MPN). For comparison, the viable cell counts at the start and after appropriate intervals were recorded and expressed as logarithmic counts. The revived cultures were observed for mutation, change in pigmentation or colony morphology.
under various conditions
Logarithmic
counts valueb
Before
After storage forh Weeks
Months
1
3
6
12
18
6
12
20
A” B” C”
6 8 8
0 1 7
0 5 5
NT 5 5
NT 4 5
NT 3 5
NT 1 5
NT NT 4
Chlorojlexus aurantiacur DSM 636
A B C
I 8 7
5 7 6
3 8 I
0 7 6
NT 7 6
NT 4 6
NT 1 6
NT NT 4
Chlorofzexus aggregans DSM 9485
A B C
6 I 7
5 7 7
0 7 7
NT 6 6
NT 5 6
NT 2 5
NT 1 4
NT NT NT
Chlorojexus DSM 635
aurantiacuy
NT, Not tested. a Equal volumes of conventional and modified media were inoculated with an equal volume of inoculum from freshly grown cultures. For storage, the cultures were prepared according to three different methods (A. B, C) and were kept under parallel conditions. For more details, see Section 2. b After storage, serial decimal dilutions were prepared in duplicate. The highest dilution n to yield visible growth gave total viable counts of 10” cells ml-’ (or n log when expressed as logarithmic counts). The results were thus expressed as average colony-forming units per sample.
154
K.A. Malik I Journal of Microbiological
3. Results and discussion Pure liquid cultures of Chlorojkxus were stored successfully with this method for several months without subculturing. Throughout this period, the stored cultures with additives (Table 2, B and C) showed normal growth on reinoculation into fresh medium within a short time. During storage, a regular viability assay was conducted after set intervals which showed a good survival rate. The anaerobic conditions and the presence of sulfide in the modified medium (Table 1) favoured growth and prolonged viability during maintenance as slow growing liquid cultures. Neutralized sodium sulfide solution, which was injected to lower the redox potential, proved effective as Chlorojlexus apparently also grows photoautotrophically with sulfide as electron donor [l-4,12]. Under such reduced conditions, the liquid cultures could be viably maintained as slow growing cultures for several months at 37-40°C in dim light (100-200 lx) when they were regularly supplemented (at least once a month) with yeast extract (Table 2, B and C). However, total lysis was observed within a few days during storage under conventional semiaerobic conditions and the cultures died more quickly when maintained at normal growth temperature and light intensity (Table 2, A). During preliminary experiments, the comparative effect of various other maintenance conditions (no light, low maintenance temperature of 4-25°C) and growth supplements (such as glycyl-glycine, acetate) was also studied. However, this did not prove effective prolonging the subculturing interval and the viability of liquid cultures during long-term maintenance [4]. The accumulation of waste products causes deterioration and damage to living cells during longterm maintenance as standing liquid cultures. The viability of various standing cultures could be extended by the addition of adsorbents or stabilizers [4,13,14]. By increasing the span of generation time, various sensitive cell cultures of plants and unicellular green algae have already been maintained successfully for several months as slow growing cultures [13,14]. The use of activated charcoal has already been
Methods 27 (1996) 151-155
recommended by the author for the prolonged maintenance of sensitive active living cell suspensions as it absorbs various gases, free radicals, harmful metabolites and waste products [4,13,15]. In the case of ChZorojGxus aurantiacus, all the tested cultures also showed good survival recoveries and after more than 20 months of storage only a slight drop in their viability counts occurred in the presence of activated charcoal (Table 2, C). Such maintained cultures at slow growth rate, proved stable and cells appeared healthy for extended storage (Table 2, C). However, activated charcoal should not be added at the start of growth as it slows this down. During the initial stages, the samples were regularly supplemented after every 2 weeks with yeast extract (0.1% end concentration). However, it was later found to be unnecessary and feeding at a monthly interval proved satisfactory. In the maintained cultures, no change in pigmentation or colony morphology was observed, indicating that the maintenance and cultivation conditions were optimal for photoautotrophic growth. The liquid cultures tested (Table 2, C) proved viable even beyond 20 months of storage. However, proper viability assay and the regrowth experiments could not be conducted beyond this time due to the decrease in volume of cell suspensions caused by periodic sampling. Although some loss in viability of cells was observed after several months of maintenance, the final concentration of living cells was high enough to provide an adequate source of inoculum for routine use. Using this method, the cultures of newly described Chlorojkxus aggregans [lo] were also successfully maintained for several months as slow growing liquid cultures (Table 2, C). Thus the storage of sensitive Chlorojexus for long time periods as slow growing standing cultures is an attractive advantage, since the cultures so preserved can be stored without periodic shaking and transfer to fresh medium. No further attention was required during storage. Due to the use of moderate conditions, regrowth of the cultures was faster than the reactivation from dried or cryopreserved preparations where cultures are mostly subjected to very drastic conditions. The method is convenient for routine work as the maintained cell suspensions can provide a continuous
K.A. Malik I Journal of Microbiological Methods 27 (1996) 1.51- 155
source of inocula, from suspension, for immediate
the same use.
batch
of cell [7]
Acknowledgments [8]
I am grateful assistance.
to Andrea
Schiitze
for technical
[9]
References [lo] Castenholz, R.W. and Pierson, B.K. (1981) Isolation of members of the family Chloroflexaceae. In: The Prokaryotes (Eds. M.P. Starr, H. Stolp, H.G. Triiper, A. Balows and H.G. Schlegel). Springer, Berlin, pp. 290-298. PI Pierson, B.K and Castenholz, R.W. (1992) The family Chloroflexaceae. In: The Prokaryotes (Eds. A. Balows, H.G. Triiper, M. Dworkin. W. Harder and K.H. Schleifer), Second Edition. Springer, Berlin, pp. 3754-3774. [31 Malik, K.A. (1983) A modified method for the cultivation of phototrophic bacteria. J. Microbial. Methods 1, 343-352. bacteria. [41 Malik, K.A. (1991) Maintenance of phototrophic In: Maintenance of Microorganisms and Cultured Cells (Eds. B.E. Kirsop and A. Doyle). Academic Press, London, pp. 82-100. photo[51 Pfennig, N. and Triiper, H.G. (1989) Anoxygenic trophic bacteria. In: Bergey’s Manual of Systematic Bacteriology, Vol. 3 (Eds. J.T. Staley, M.P. Bryant, N. Pfennig and J.G. Holt). Williams and Wilkins, Baltimore, pp. 16351709. [cl Castenholz, R.W. (1’389) Genus ChioroJlexus. In: Bergey’s Manual of Systematic Bacteriology, Vol. 3 (Eds. J.T. Staley.
[ 111
[ 121
[ 131
[14]
[15]
155
M.P. Bryant, N. Pfennig and J.G. Holt). Williams and Wilkins, Baltimore, pp. 1698-1709. Van Niel, C.B. (1971) Techniques for the enrichment, isolation and maintenance of the photosynthetic bacteria. In: Methods in Enzymology,Vol. 23, Part A (Ed. A. San Pietro). Academic Press, New York, pp. 2-3. Schmidt, K. (1980) A comparative study on the composition of chlorosomes (Chlorobium vesicles) and cytoplasmic membranes from Chlorojlexus auranfiacus strain OK-70-fl and Chlorobium limicola f. thiosulfatophilum strain 6230. Arch. Microbial. 124, 21-31. Malik, K.A. (1996) A modified medium and method for the cultivation of Chlorojlexus. J. Microbial. Methods 27, 147150. Hanada, S., Hiraishi, A., Shimada, K. and Matsura, K., (1995) Chlorojlexus aggregans sp. nav., a phototrophic filamentous bacterium which forms dense aggregates by active gliding movement. Int. J. Syst. Bacterial. 45, 676681. Malik, K.A. (1992) Some universal media for the isolation. growth and purity checking of a broad spectrum of microorganisms. World J. Microbial. Biotechnol. 8, 453-456. Madigan, M.T. and Brock, T.D. (1975) Photosynthetic sulfide oxidation by Chlorojexus auranriacus, a filamentous photosynthetic, gliding bacterium. J. Bacterial. 122, 782784. Malik, K.A. (1995) A convenient method to maintain unicellular green algae for long times as standing liquid cultures. J. Microbial. Methods 22, 221-227. Schumacher, H.M.. Malik, K.A. and Van hen, F. (1995) Simple storage of plant cell cultures in liquid media. World J. Microbial. Biotechnol. 11, 703-705. Malik, K.A. (1990) Use of activated charcoal for the preservation of anaerobic phototrophic and other sensitive bacteria by freeze-drying. J. Microbial. Methods 12, 117124.
Journal ofMicrobiological Methods
Journal of Microbiological Methods 27 ( 1996) 15I- 155
A convenient method for maintaining ChZoroJexus for long time periods as slow growing liquid cultures Khursheed
A. Malik*
DSMZ-Deutsche Sammlung van Mikroorganismen und Zellkulturen GtnbH, Mascheroder Weg lb, 38214 Braunschweig. Germany
Received 12 August 1996; accepted 19 August 1996
Abstract A simple procedure is described to maintain Chlor-&xus cultures for relatively long times without frequent subculturing. All tested strains of CJzlorojexus auruntiucus proved viable after more than 18 months of storage in dim light (100-200 lx)
at 37-40°C as slow growing standing liquid cultures. During storage, no shaking, regular subculturing or transfer to fresh medium was required. To increase the span of generation time, cell survival and stability, various growth components and adsorption materials were screened. The anaerobic conditions, presence of sulfide and regular feeding with yeast extract favored prolonged viability during maintenance as liquid cultures. The presence of activated charcoal (0.05% w/v) in the media resulted in slowing down the growth rate and due to the absorption of harmful metabolites and waste products, the cells remained more rstable and healthy during such maintenance. The cultures of Chloroflexus aggregans also survived several months under such conditions. Due to the use of moderate conditions, regrowth in fresh medium resulted within relatively short times with full pigmentation, indicating that the maintenance and cultivation conditions were optimal for photoautotrophic growth. The method is more suitable, effective and convenient than cryopreservation or lyophilization as the maintained cell suspensions can provide a continuous source of inocula, from the same batch of cell suspension, for routine work. Keywords:
ChZoroJlexm;
Slow growing;
Standing liquid cultures; Long-term
1. Introduction Difficulties in the cultivation and laboratory maintenance of phcltosynthetic flexibacteria Chloroflexus are well known [ 1,2]. Liquid pure cultures of Chlorojkxus are dlifficult to maintain at normal growth temperature, and light intensity. Although media and methods for their culturing have improved greatly over the past few years, their cultivation and maintenance still causes problems [3-61. Chloroplexus aurantiacus, the first genus and species of the *Fax: +49 531 2616418.
Adsorption
materials
family Chloroflexaceae is generally maintained as living culture with a bi-weekly subculturing interval involving a frequent shaking. To avoid frequent transferring, the use of a co-culture (by mixing the culture with a known pure strain of cyanobacterium) has also been reported [ 1,7]. However, to retrieve a pure culture, a cumbersome reisolation is required every time. Frequent regular subculturing of stocks has many disadvantages and by increasing the transfer interval, costly and labour intensive frequent serial subculturing can be avoided. The effective maintenance of Chlorojkus is thus desirable so that the maintained
0167.7012/96/$15,00 Copyright 0 1996 Elsevier Science B.V. All rights reserved PZZ SO167-7012(96)00942-6
maintenance;
K.A. Mulik I Jourtml oj Microbiologicul
152
cell suspensions are immediately available as a continuous source of inoculum. Some preliminary results showing the comparative effect of various maintenance conditions and several growth supplements on long-term maintenance of such liquid cultures have already been reported [3,4]. This study presents data on the successful maintenance of ChloroJexus aurantiacus and Chloroflexus aggregans for long time periods as slow growing living cultures without frequent subculturing.
2. Material
and methods
2. I. Microorganisms Four strains of Chlorojlexus aurantiacus (DSM 635, 636, 637 and 638) and two strains of Chloro@ems aggregans (DSM 9485 and 9486) were used throughout this work. All bacteria were from the German Collection of Microorganisms and Cell Cultures (DSMZ). 2.2. Preparation
of cultures
For maintenance, the cultures were grown under semiaerobic conditions (in lOO-ml flat glass bottles with screw-caps) in the conventional Medium 1 (described by Schmidt [g]) and under anaerobic conditions (in lOO-ml flat glass bottles with screw caps with holes for the injection of the samples and butyl rubber septa) in a modified Medium 2 (described by the author in detail [9], and listed in Table 1). For Chlorojlexus aggregans, 1.0 ml/l, sterile vitamin solution CA [lo] was added to Medium 1 and 2. The cultures were incubated at 50°C at a light intensity of 300-500 lx. 2.3. Preparation and additives
of growth components,
adsorbents
Solutions of yeast extract (10% w/v kept under N?) and 10.0% (w/v) neutral activated charcoal (from Caelo, 4010 Hilden, FRG or any other of medicinal grade) were prepared in distilled water, autoclaved at 115°C for 13 min and stored at 4°C. Neutralized sodium sulfide solution (1.5% w/v in H,O) was prepared in a closed screw-capped bottle
Methods -77 (1996) 151-155
Table I Modified Growth Medium 2 for Chloroj7aus Yeast extract 1.0 g 1.0 g Glycyl-glycine NazHP0,X2H20 0.1 g 0.1 g MgSO,X7H,O 0.1 g KNO, 0.5 g NaNO i 0.1 g NaCl 0.05 g CaClzX2Hz0 5.0 ml Ferric citrate solution (0.1 g in 100 ml HZO) I .O ml Trace element solution SL-6 1050.0 ml Distilled water The pH of this medium was adjusted to 8.2. The medium was boiled under a stream of N2 for a few minutes and 90 ml medium was distributed into 100.ml screw-capped bottles with screw caps having holes and butyl tubber septa. Each bottle was bubbled with N1 and closed immediately with a rubber septum and screw tightened. The bottles were autoclaved at 121°C for 15 min. After autoclaving. 1.0 ml of neutralized sulfide solution (0.015% end concentration [3]) was injected into each bottle. For Chforojlexus nggregam, 1.O ml/l, sterile vitamin solution CA [IO] was added. This medium can be stored for several months under anaerobic conditions. Trace element solution SL-6: ZnSO,X7H,O MnCI,X4HZ0 HISO, CoCl,x6H,O CuCI,X2H,O NiC12X6H,0 Na,MoO,X2H,O Distilled water
0.1 g 0.03 g 0.3 g 0.2 g 0.01 g 0.02 g 0.03 g 1000 ml
with a butyl rubber septum and pH was adjusted to about 7.3 by adding 2 M H,SO, drop-wise with a syringe without opening the bottle [3]. 2.4. Storage of liquid cultures For comparison, stock cultures were grown to the logarithmic growth phase in different media under three different conditions and were kept for storage under parallel conditions. A: Control, with no additives. Kept under semiaerobic conditions after growing in Medium 1 [S]. B: No additives. Kept under anaerobic conditions in the presence of sulfide after growing in modified Medium 2 [9]. C: Supplemented with 0.05% activated charcoal after good growth appeared in modified Medium 2. The addition of activated charcoal was avoided at the beginning as it slows down the growth. Cultures were kept
K.A. Malik I Journal of Microbiological
under anaerobic conditions in the presence of sulfide and activated charcoal. All the cultures were maintained under parallel conditions for more than 20 months at 37-40°C in dim light (loo-200 lx). The samples were regularly supplemented every 2 weeks (for the first 3 months) and later at monthly intervals (for 12-20 months) with yeast extract (0.1% end concentration using a l-ml syringe flushed with nitrogen gas). For viability assay from the maintained culture, 1.0 ml was withdrawn (using a sterile gas-tight 2-5 ml syringe) at regular intervals and serial decimal dilutions were prepared. After showing non-viability, the culture A (Table 2) was not supplemented further. For viability assay from the maintained cultures, 1.0 ml was withdrawn at regular intervals and serial decimal dilutions were prepared. 2.5. Recultivation
ofcultures and sterility test
For recultivation, about 1 ml of the stored cell suspension was transferred to 14 ml of fresh medium (in Hungate tube). The standing cultures were replenished with equal amounts of fresh medium. For sterility tests, cultures were streaked on normal agar medium plates and on a universal heterotrophic Table 2 Survival of Chlorof7exu.r liquid cultures
during long term maintenance
Media and conditions”
Strain
Methods 27 (1996) 151-155
153
medium H,P [ 111. The cultures were grown at 49°C under light (500-1000 lx).
2.6. Viability assay The viability assay was done first after every 3 weeks, later every 6 weeks and finally after every 6 months of storage. For precise determination of viable cell counts during storage, DSM 635, DSM 636 and DSM 9485 were selected. For the estimation of viable cell counts, 0.5 ml of inocula was transferred from the standing cultures (for reproducible results, a through mixing of the dilution tubes was necessary to eliminate clumps) and injected into 4.5 ml of prereduced medium in screw-cap tubes (Hungate tubes with septa, Bellco Glass Inc., 204716125) and 6-8 serial decimal dilutions were prepared using oxygen free syringes with incubation under appropriate conditions. The number of viable cells was determined using the most probable number method (MPN). For comparison, the viable cell counts at the start and after appropriate intervals were recorded and expressed as logarithmic counts. The revived cultures were observed for mutation, change in pigmentation or colony morphology.
under various conditions
Logarithmic
counts valueb
Before
After storage forh Weeks
Months
1
3
6
12
18
6
12
20
A” B” C”
6 8 8
0 1 7
0 5 5
NT 5 5
NT 4 5
NT 3 5
NT 1 5
NT NT 4
Chlorojlexus aurantiacur DSM 636
A B C
I 8 7
5 7 6
3 8 I
0 7 6
NT 7 6
NT 4 6
NT 1 6
NT NT 4
Chlorofzexus aggregans DSM 9485
A B C
6 I 7
5 7 7
0 7 7
NT 6 6
NT 5 6
NT 2 5
NT 1 4
NT NT NT
Chlorojexus DSM 635
aurantiacuy
NT, Not tested. a Equal volumes of conventional and modified media were inoculated with an equal volume of inoculum from freshly grown cultures. For storage, the cultures were prepared according to three different methods (A. B, C) and were kept under parallel conditions. For more details, see Section 2. b After storage, serial decimal dilutions were prepared in duplicate. The highest dilution n to yield visible growth gave total viable counts of 10” cells ml-’ (or n log when expressed as logarithmic counts). The results were thus expressed as average colony-forming units per sample.
154
K.A. Malik I Journal of Microbiological
3. Results and discussion Pure liquid cultures of Chlorojkxus were stored successfully with this method for several months without subculturing. Throughout this period, the stored cultures with additives (Table 2, B and C) showed normal growth on reinoculation into fresh medium within a short time. During storage, a regular viability assay was conducted after set intervals which showed a good survival rate. The anaerobic conditions and the presence of sulfide in the modified medium (Table 1) favoured growth and prolonged viability during maintenance as slow growing liquid cultures. Neutralized sodium sulfide solution, which was injected to lower the redox potential, proved effective as Chlorojlexus apparently also grows photoautotrophically with sulfide as electron donor [l-4,12]. Under such reduced conditions, the liquid cultures could be viably maintained as slow growing cultures for several months at 37-40°C in dim light (100-200 lx) when they were regularly supplemented (at least once a month) with yeast extract (Table 2, B and C). However, total lysis was observed within a few days during storage under conventional semiaerobic conditions and the cultures died more quickly when maintained at normal growth temperature and light intensity (Table 2, A). During preliminary experiments, the comparative effect of various other maintenance conditions (no light, low maintenance temperature of 4-25°C) and growth supplements (such as glycyl-glycine, acetate) was also studied. However, this did not prove effective prolonging the subculturing interval and the viability of liquid cultures during long-term maintenance [4]. The accumulation of waste products causes deterioration and damage to living cells during longterm maintenance as standing liquid cultures. The viability of various standing cultures could be extended by the addition of adsorbents or stabilizers [4,13,14]. By increasing the span of generation time, various sensitive cell cultures of plants and unicellular green algae have already been maintained successfully for several months as slow growing cultures [13,14]. The use of activated charcoal has already been
Methods 27 (1996) 151-155
recommended by the author for the prolonged maintenance of sensitive active living cell suspensions as it absorbs various gases, free radicals, harmful metabolites and waste products [4,13,15]. In the case of ChZorojGxus aurantiacus, all the tested cultures also showed good survival recoveries and after more than 20 months of storage only a slight drop in their viability counts occurred in the presence of activated charcoal (Table 2, C). Such maintained cultures at slow growth rate, proved stable and cells appeared healthy for extended storage (Table 2, C). However, activated charcoal should not be added at the start of growth as it slows this down. During the initial stages, the samples were regularly supplemented after every 2 weeks with yeast extract (0.1% end concentration). However, it was later found to be unnecessary and feeding at a monthly interval proved satisfactory. In the maintained cultures, no change in pigmentation or colony morphology was observed, indicating that the maintenance and cultivation conditions were optimal for photoautotrophic growth. The liquid cultures tested (Table 2, C) proved viable even beyond 20 months of storage. However, proper viability assay and the regrowth experiments could not be conducted beyond this time due to the decrease in volume of cell suspensions caused by periodic sampling. Although some loss in viability of cells was observed after several months of maintenance, the final concentration of living cells was high enough to provide an adequate source of inoculum for routine use. Using this method, the cultures of newly described Chlorojkxus aggregans [lo] were also successfully maintained for several months as slow growing liquid cultures (Table 2, C). Thus the storage of sensitive Chlorojexus for long time periods as slow growing standing cultures is an attractive advantage, since the cultures so preserved can be stored without periodic shaking and transfer to fresh medium. No further attention was required during storage. Due to the use of moderate conditions, regrowth of the cultures was faster than the reactivation from dried or cryopreserved preparations where cultures are mostly subjected to very drastic conditions. The method is convenient for routine work as the maintained cell suspensions can provide a continuous
K.A. Malik I Journal of Microbiological Methods 27 (1996) 1.51- 155
source of inocula, from suspension, for immediate
the same use.
batch
of cell [7]
Acknowledgments [8]
I am grateful assistance.
to Andrea
Schiitze
for technical
[9]
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