A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions

Journal o f Microbiological Methods 2 (1984) 41-47

41

Elsevier JMM 00044

A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions K.A. M a l i k Deutsche Sammlung yon Mikroorganismen, Gesellschaft far Biotechnologische Forschung mbH, Grisebachstrasse 8, D-3400 GOttingen (F. R. G. )

(Received 8 July 1983) (Revised version received 4 October 1983) (Accepted 7 October 1983)

Summary A simple, effective and economical method for the long-term preservation of bacteria in liquid nitrogen under anaerobic conditions is described. As a case example anaerobic photosynthetic bacteria were successfully preserved. Gas tight small screw-cap glass ampoules with butyl rubber septa were used for freezing the specimen anaerobically. During experimental manipulations no anaerobic chamber or glove boxes were required. All tested cultures yielded high recoveries after repeated thawing and during storage. After freezing, survival recoveries of Rhodospirillaceae ranged from 70-100%, whereas with strict anaerobic strains of Chlorobiaceae and Chromatiaceae a maximum loss of 1-2 Iogm0counts was observed. No further loss in viability occurred after 1-2 years of storage. The small size of the ampoules and the use of single ampoule for 15-20 repeated retrievals proved economical with respect to storage space and costs. The system is compact and suitable for the preservation of anaerobic phototrophie bacteria and other fragile anaerobic microorganisms. Key words: Anaerobic conditions - Long term preservation - Liquid nitrogen - Phototrophic bacteria

Introduction It is g e n e r a l l y d e s i r e d t h a t s t o c k c u l t u r e s s h o u l d be p r e s e r v e d u n d e r such c o n d i tions t h a t e n s u r e viability, p r e v e n t c o n t a m i n a t i o n a n d m i n i m i z e g e n o t y p i c a n d phenotypic variations. A d v a n t a g e s o f liquid n i t r o g e n f o r p r e s e r v a t i o n o f m i c r o o r g a n i s m s h a v e b e e n e m p h a s i z e d [1-7]. F o r effective p r e s e r v a t i o n u n d e r a n a e r o b i c c o n d i t i o n s , a n d to y i e l d high r e c o v e r i e s o f a n a e r o b i c b a c t e r i a a f t e r s t o r a g e in liquid n i t r o g e n , e x p e r i m e n t a l m a n i p u l a t i o n s a r e p e r f o r m e d g e n e r a l l y w i t h i n an a n a e r o b i c c h a m b e r , o r 0167-7012/84/$03.00 © 1984 Elsevier Science Publishers B.V.

42 else cumbersome methods like heat sealing of glass ampoules under a stream of nitrogen gas are employed [7, 8]. Among phototrophic bacteria all forms of green sulfur bacteria (Chlorobiaceae) and purple sulfur bacteria (Chromatiaceae) are strictly anaerobic. Although purple non-sulfur bacteria (Rhodospirillaceae) are generally microaerophilic, strict anaerobic conditions are required for their normal photoautotrophic growth [9]. For the preservation of phototrophic bacteria in liquid nitrogen it is also essential that anaerobic conditions should be assured. Long term preservation of phototrophic bacteria in liquid nitrogen under conventional aerobic conditions using sealable glass or polypropylene ampoules, and under semi-anaerobic conditions using polypropylene screw-cap ampoules, has previously been reported [10, 11]. Due to low viability, retrieval of strictly anaerobic phototrophic bacteria was complicated and time consuming when such, and other similar, methods were used for liquid nitrogen storage [10-13]. This paper describes a compact, simple, effective and economical method for liquid nitrogen storage and retrieval of phototrophic bacteria under anaerobic conditions. Material and Methods

Organisms and growth conditions All bacteria used were strains from the Deutsche Sammlung von Mikroorganismen (DSM). A modified method previously described [9] for the cultivation of phototrophic bacteria was used to grow the strains photoautotrophically. During experimental manipulations no anaerobic chamber was required and the anaerobiosis was generally maintained as described by Hungate [14, 15].

Equipment For freezing of bacterial suspensions, small screw-cap glass ampoules of 2 ml capacity (10 x 30 mm) were used. These were obtained from Varian GmbH, Darmstadt, F.R.G., and are generally used for 'Autosampler' in gas chromatography. The ampoules were provided with red rubber septa and plastic (autoclavable) screw-caps with holes at the centre for injection of samples. The red rubber septa were replaced with oxygen-impermeable butyl rubber septa. The ampoules were washed, rinsed with distilled water, tightly closed and autoclaved. Before use these were labelled with the numbers of the strains to be preserved and, using a sterile gas-tight 5-10 ml syringe, these were evacuated to facilitate filling. For freezing in liquid nitrogen aluminium canes or holders were used, to which ampoules were clamped.

Cryoprotective agent Dimethylsulfoxide (DMSO) was used as a cryoprotectant. DMSO solution was sterilized by autoclaving at 114°C for 10 rain or it was sterilized by filtration.

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Preparation of cell suspension for freezing Cultures were grown photoautotrophically until the mid-to-late logarithmic phase of growth [9]. For harvesting, the cultures were centrifuged for 30 min at 4000 x g in the screw-cap bottles in which they were grown. Supernatant was decanted with a long needle syringe without opening the bottle. To facilitate removal, and to maintain anaerobiosis, a second syringe filled with sterile nitrogen gas was inserted simultaneously into the septum. The pellets were resuspended carefully in ice cold sterile DMSO solution (5% v/v in H20 ). After being suspended in DMSO, the cells were allowed to equilibrate with the cryoprotectant for 15 min in an ice bath. For halophilic or marine bacteria the DMSO solution was supplemented with sterile NaC1 solution to obtain a final concentration of NaCI as in the growth medium.

Freezing procedure With a syringe, 1.5 ml of heavy cell suspension (108-101° cells/ml) were injected into each ampoule. The ampoules were immediately clamped onto the labelled aluminium cane, which was placed in a cannister and frozen by direct immersion in liquid nitrogen, or in the gas phase of liquid nitrogen.

Recovery of frozen suspension The frozen ampoules were removed from liquid nitrogen. For partial thawing these were immediately immersed to the neck in water baths (20-30 × 20-33 mm) at 37°C for few seconds. After thawing, the outer surfaces of the ampoules were wiped dry. With a syringe, which was already flushed and filled with sterile nitrogen gas, a small volume (about 0.05 ml) of inocula was withdrawn and injected into 5-10 ml liquid growth medium in screw-cap tubes. The rest of the cell suspension in the ampoule was immediately frozen again in liquid nitrogen for later use. The DMSO in the inocula was 100-200 times diluted in the culture medium to a non-inhibitory concentration. For green and purple sulfur bacteria, overnight incubation at low light intensity of 100--200 Ix was carried out before normal incubation [9].

Estimation of viability Survival recoveries were checked before freezing, immediately after freezing and after 1-2 years of storage. From 0.5 ml inocula, serial decimal dilutions were prepared in screw-cap tubes containing 4.5 ml medium [9]. In the case of Rhodospirillaceae, 0.1 ml volumes were withdrawn from the serial dilution and plated in duplicate [9]. The number of colonies were counted from the plates and average colony forming units per sample were calculated. In the case of green and purple sulfur bacteria serial decimal dilutions were prepared in duplicate, and the tubes were placed for growth in light chambers [9]. The highest dilution n to yield visible growth, from an average of 5 cells (which may actually vary from 1 to 9 cells), gave total viable counts of 10~ cells/ml (or n log10 when expressed as logarithmic counts). The losses of viability were thus expressed as log10 value reductions.

44 Results and Discussion

Previously it was observed that, particularly with anoxygenic green sulfur bacteria, purple sulfur bacteria and brown non-sulfur bacteria, strict anaerobic conditions resulted in improved phototrophic growth, and allowed better survival during maintenance for longer periods [9]. The procedure developed here for liquid nitrogen storage of such bacteria under anaerobic conditions complements this. Storage in gas-tight, small screw-cap glass ampoules proved relatively simple, safe, convenient and assured anaerobiosis without the use of an anaerobic chamber. During 1-2 years of use there were no blow outs or explosions during thawing and retrieval. The overall procedure is time saving and inexpensive. It can prove equally useful for small laboratories or a large culture collection. Several phototrophic bacteria which were freeze'stored 8-9 years ago in heatsealed 2 ml plastic ampoules in liquid nitrogen, using DMSO as a cryoprotectant, are still viable [11]. However, due to the use of aerobic conditions during experimental manipulations, low viability resulted, especially in the case of brown Rhodospirillaceae, Chlorobiaceae and Chromatiaceae, which are known to be difficult to preserve. Due to poor survival after freezing, retrieval and propagation of several anoxygenic phototrophic bacteria were often time consuming and not very practicable for a service culture collection. In conventional methods, for anaerobic freezing it is essential that heat-sealing of glass or plastic ampoules should be done under a stream of nitrogen gas or within an anaerobic chamber, and that similar processes are carried out during anaerobic recovery. This is however, cumbersome, expensive and time consuming. During early stages of these studies the problem was partly solved by the use of 2 ml polypropylene screw-cap ampoules with silicon gaskets [10]. Semianaerobic conditions could be assured during filling and removing the specimen under a stream of nitrogen gas. Such ampoules proved economical and could be used for repeated retrievals as compared to the heat-sealable glass or polypropylene ampoules which were used only once. Although several strains of purple non-sulfur bacteria could be stored successfully, poor viability remained a problem with strictly anaerobic bacteria [10]. With this new method, which is based on a completely closed anaerobic system, all phototrophic bacteria, including oxygen sensitive Chlorobiaceae, Chrornatiaceae, and brown Rhodospirillaceae, were successfully preserved in liquid nitrogen resulting in good viability. The viability of a few selected Rhodospirillaceae after freezing and storage in liquid nitrogen, are shown in Table 1. The results show that all tested strains yielded high recoveries after freezing. Viable plate counts yielded rapid photoautotrophic growth (2-3 days) with full pigmentation, indicating that bacteria were frozen and revived anaerobically in a healthy state. From 30 strains of Chlorobiaceae and Chromatiaceae which were successfully preserved using this method only a few selected strains were used for precise viability control (Table 2). The results after freezing, expressed in Iogi0 reductions,

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showed maximum loss of 1-2 log10 counts in almost all cases. This represents a good survival recovery, as much higher losses usually occur during freezing of similar fragile bacteria. With strains of Chloroflexus aurantiacus a maximum loss of about 2 lOgl0 counts was also observed after freezing, and relatively rapid growth occurred during retrieval when modified anaerobic cultivation conditions were used [9]. In almost all cases, viability counts after 1-2 years of storage remained practically the same as immediately after freezing in liquid nitrogen (Tables 1 and 2). Therefore, viability controls were not performed after regular intervals during further storage, as it has been observed that if an organism survives the stresses

TABLE

1

VIABILITY OF SOME LIQUID NITROGEN

RHODOSPIRILLACEAE

AFTER

FREEZING

AND

STORAGE

IN

Viable cell counts a Species

D S M No.

Before freezing

After freezing

Storage ( 1 - 2 years)

Rhodocycluspurpureus c Rhodomicrobiurn vannielii

168 162 2342 137 2131 1710 156 1709 149 161 123 130 158 159 1374 133 134 113 120 122 1774 467 107 2132 109

2.5 × 1.8 x 2.0 x 9.0 × 2.8 × 4.5 x 4.5 × 2.5 x 5.0 x 5.0 x 2.5 x 1.0 x 7.5 x 2.5 x 1.0 x 1.5 x 2.0 x 7.5 x 3.5 x 1.0 x 1.0 x 1.5 x 3.5 x N.T. 1.2 x

2.1 × 1.5 x 1.2 × N.T. 2.3 × 2.5 × 2.5 x 1.6 x 3.5 x 6.0 x 2.3 x 8.5 x 6.0 x 1.6 × 4.1 × 8.5 x 1.5 x 5.0 x 2.5 x N.T. N.T. 2.0 x 2.5 x 6.0 x 6.5 x

N.T. b N.T. 1.0 x 101° 3.0 × 107 2.5 × 10 l° 5.0 × 10 l° 4.0 x 109 2.5 x 10 l° 4.5 x 101° 2.5 x 108 2.3 x 109 8.0 x 10 l° 4.0 x 108 7.5 × 109 7.2 x 109 5.4 x 108 1.1 x 108 3.8 x 107 1.5 x 108 2.0 x 106 5.0 x 106 1.0 x 108 3.0 x 10 I° 5.0 x 108 3.1 x 108

Rhodopseudomonas acidophila R. blastica R. capsulata R. gelatmosa R. globiformis R. palustris R. sphqeroides R. sulfidophila c R. viridis c Rhodospirillumfulvum c R. molischianum c R. photometricum c R. rubrum R. salexigenes R. tenue a b c

106 108 10 l0 107 101° 101° 109 10 l° 10 l° 108 109 10 ll 108 101° 101° 108 108 10;' 108 107 107 t08 101° 109

106 108 10 l° 101° 10 l° 109 101° 10 I° 108 109 10 I° 108 1010 109 108 108 107 108

108 10 m 106 101°

The numbers indicate average colony forming units per sample. For details see Material and Methods. Not tested. Strict anaerobic conditions are essential for photoautotrophic growth and during maintenance [9].

46 TABLE 2 L O G A R I T H M I C C O U N T S ON SOME C H L O R O B I A C E A E F R E E Z I N G A N D S T O R A G E IN L I Q U I D N I T R O G E N

AND C H R O M A T I A C E A E

AFTER

Logarithmic counts value d Species a

DSM No.

Before freezing

After freezing

Storage (1-2 years)

Amoebobacter roseus Chlorobium limicola Chlorobium vibrioforme Chromatium gracile Chromatiurn minutissimum Chromatium vinosum Chromatiurn violascens Chromatium warmingii Ectothiorhodospira halochloris Ectothiorhodospira halophila Ectothiorhodospira shaposhnikovii Thiocapsa roseopersicina Thiocapsa pfennigii

235 245 263 203 1367 180 198 175 1059

5 6 6 7 7 7 5 5 7

4 5 5 6 7 N.T. 3 N.T. 6

3 5 N.T. c N.T. 6 6 3 3 N.T.

244

6

6

6

243 217 226 208

6 7 6 5

5 5 5 3

5 5 4 3

Thiocystis violacea

b c

All forms are strictly anaerobic under phototrophic conditions. For details see Material and Methods. Not Tested.

of freezing and thawing then it generally remains viable indefinitely, and no further significant loss of viability occurs. Each ampoule is used for several repeated retrievals. Although withdrawal of inocula from the same ampoule over a period of time is advantageous, repeated thawing and refreezing results, normally, in D N A damage and loss of viability if a good cryoprotectant is not used [4, 16]. To overcome this problem, an effective cryoprotectant (DMSO) was used, and rapid and partial thawing of the ampoules was done during repeated retrievals. In no case was complete thawing of the ampoules needed, as in each case a small amount (about 0.05 ml) of partially thawed suspension was withdrawn as inocula, and this proved enough for reasonable growth in 5-10 ml of liquid medium within a short time. During 1-2 years of storage all tested strains proved viable after several repeated freeze-thawings. Economy of storage in liquid nitrogen has often been emphasized [1, 4, 6, 17]. To overcome the space problem in deep-freezers and in liquid nitrogen, generally small glass beads and plastic or glass capillary tubes have been used [3, 4, 8, 10, 13, 18]. Although such miniaturized methods are useful for aerobic microorganisms, an anaerobic chamber or continuous gassing with nitrogen is required to maintain anaerobiosis for processing of anaerobes. In mini screw-cap glass ampoules it became possible to easily store specimens under strict anaerobic conditions without such arrangements. The small size of the ampoules, and the use of

47

a single ampoule for 15-20 repeated retrievals, proved economical with respect to storage space and costs. The results of high survival recoveries and rapid photoautotrophic growth indicate that the procedure used to freeze and revive is most suitable for anaerobic phototrophic bacteria, and that it should be equally applicable to other anaerobic microorganisms. References 1 2 3 4 5

6 7 8

9 10 11

12 13 14 15 16 17 18

Jarvis, J.D., Wynne, C.D. and Teller, E.R. (1967) Storage of bacteria in liquid nitrogen. J. Med. Lab. Technol. 24, 312-314. Swoager, W.C. (1972) Preservation of microorganisms by liquid nitrogen refrigeration. Am. Lab. 4, No. 12, pp. 45-52. Nagel, J.G. and Kunz, J. (1972) Simplified storage and retrieval of stock cultures. Appl. Microbiol. 23, 837-838. Daily, W.A. and Higgens, C.E. (1973) Preservation and storage of microorganisms in the gas phase of liquid nitrogen. Cryobiology 10, 364-367. Dietz, A. (1975) Nitrogen preservation of stock cultures of unicellular and filamentous microorganisms. In: Round Table Conference on the Cryogenic Preservation of Cell Cultures (Rinfret, A.P. and La Salle, B., eds.), pp. 22-36. National Academy of Sciences, Washington, D.C. Rinfret, A.P. and Lasalle, B. (1975) Round Table Conference on the Cryogenic Preservation of Cell Cultures. National Academy of Sciences, Washington, D.C. Simione, F.P. and Daggett, P.M. (1977) Recovery of a marine dinoflagellate following controlled and uncontrolled freezing. Cryobiology 14, 362-366. (3ilmour, M.N., Turner, G., Berman, R.G. and Krenzer, A.K. (1978) Compact liquid nitrogen storage system yielding high recoveries of Gram-negative anaerobes. Appl. Environ. Microbiol. 35, 84--88. Malik, K.A. (1983) A modified method for the cultivation of phototrophic bacteria. J. Microbiol. Methods 1,343-352. Malik, K.A. (1981) The developments in the maintenance and preservation of phototrophic bacteria. In: Fourth Int. Conference on Culture Collections, p. 15, Abstracts, Brno. Biebl, H. and Malik, K.A. (1976) Long-term preservation of phototrophic bacteria. In: Proceedings of the Second Int. Symposium on Photosynthetic Prokaryotes (Codd, G.A. and Stewart, W.D.P., eds.), pp. 31-33, Dundee. Simione, F.P., Jr., Daggett, P., McGrath, M.S. and Alexander, M.I. (1977) The use of plastic ampoules for freeze-preservation of microorganisms. Cryobiology 141,500-502. Hippe, H., Hoffmann, P. and Malik, K.A. (1981) Capillary-tube method for freeze-preservation of microorganisms. In: Fourth Int. Conference on Culture Collections, Poster, Brno. CSSR. Hungate, R.E. (1969) A roll tube method for cultivation of strict anaerobes. Methods Microbiol. 3b, 117-132. Macy, J.M., Snellen, J.E. and Hungate, R.E. (1972) Use of syringe method for anaerobiosis. Am. J. Clin. 25, 1318-1323. Calcott, P.H. and Gargett, A.M. (1981) Mutagenicity of freezing and thawing. FEMS Microbiol. Lett. 10, 151-155. Bullen, J.J. (1975) A new technique for recovering bacteria in liquid nitrogen. J. Gen. Microbiol. 89, 205-207. Feltham, R.K.A., Power, A.K., Pell, P.A. and Sneath, P.H.A. (1978) A simple method for storage of bacteria at -76°C. J. Appl. Microbiol. 44, 313-316.