APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1976, p. 777-780 Copyright © 1976 American Society for Microbiology
Vol. 32, No. 6 Printed in U.S.A.
Viability of Lyophilized Cyanobacteria (Blue-Green Algae) LINDA L. CORBETT AND DOROTHY L. PARKER*
Biology Department, University of Wisconsin at Oshkosh, Oshkosh, Wisconsin 54901
Received for publication 11 June 1976
Lyophilization of 13 cyanobacterial cultures belonging to seven genera was attempted in a variety of suspending susbstances. All organisms survived lyophilization when suspended in lamb serum. Some of the organisms could be successfully lyophilized in horse serum, beef serum, fetal bovine serum, or human ascites fluid. With the exception of Nostoc muscorum, none of the organisms survived lyophilization in skim milk medium, egg albumin medium, or Jansen salts medium. mg; Tricine buffer (Sigma Chemical Co.), 250 mg; and sufficient 1 N NaOH to adjust the medium to pH 8.3 at 25°C. Jansen agar medium is Jansen liquid medium supplemented with 10 g of purified
Preservation of stock bacterial cultures by lyophilization is a convenient, reliable, and inexpensive procedure allowing compact storage of stock cultures (2, 6, 7). Lyophilization minimizes the hazards of genetic drift, contamination, culture death, and accidental mislabeling which can occur with routine passage. For these reasons, lyophilization is frequently the method of choice for long-term maintenance of stock cultures, when the organisms can be shown to survive the lyophilization procedure itself (2, 6, 7). Although successful lyophilization of a few species of cyanobacteria has been reported (1, 4, 5, 10), attempts to lyophilize many other cyanobacteria have failed (4, 5). Most of the cyanobacteria previously shown to survive lyophilization are filamentous organisms producing heterocysts or akinetes (spores), specialized cells which may be unusually resistant to freezing and drying. In addition, many of the lyophilizable cyanobacteria were isolated from the Antarctic (4), where frequent natural freezing and wind-produced drying may mimic lyophilization. In the present study, we have attempted to develop a lyophilization procedure that would preserve a wider variety of cyanobacteria, including several nonfilamentous and nonsporulating species isolated from the north temperate climatic zone.
agar/liter (Difco Laboratories). Suspending media for lyophilization were: skim milk medium (Difco), egg yolk enrichment medium (Difco), Jansen liquid medium, tissue culture select fetal bovine serum (Berkeley Biological Laboratories) lamb serum (Grand Island Biological Co.), beef serum (Difco), horse serum from fresh defibrinated horse blood (Grand Island Biological Co.), and human ascites fluid (Berkeley Biological Laborato-
MATERIALS AND METHODS The cyanobacteria, their origins and strain reference numbers are listed in Table 1. Jansen liquid medium contains per liter: NaNO3, 670 mg; MgSO4 -7H0, 100 mg; Na2HPO4 3H20, 17 mg; K2HPO4- 3H20, 50 mg; Na2SiO3- 5H20, 75 mg; MgCl2 6H20, 50 mg; FeCl3* 6H20, 5 mg; CaCl12 2H20, 42 mg; H3BO3, 3.25 mg; MnSO4 *4H2O, 2.4 mg; ZnSO4 7H20, 0.3 mg; (NH4)Mo71024 4H20, 0.08 mg; CuSO4-5H2O, 0.13 mg; Co(NO3)2-6H2O, 0.2 mg; KI, 0.09 mg; disodium dinitrilotetraacetate (EDTA), 4
ries). All cultures, whether in liquid or solid media, were grown without shaking at 27°C and at 1,280 lux of illumination from Westinghouse F 40-CW fluorescent lights. Before lyophilization, cultures of each organism were propagated for 10 days on the surface of Jansen agar medium in petri plates (100 by 15 mm). These cells were suspended to concentrations between 2 x 107 and 3 x 107 cells/ml by washing the agar surface with the appropriate lyophilization medium while scraping the surface with a sterile inoculation loop. A 0.5-ml portion of each cell suspension was transferred to each of several 1-ml vacuoles (Wheaton Scientific Co.), then shell frozen in propanol-dry ice. The frozen vacuoles were connected to a Virtis model 10-OlOBA lyophilizer (The Virtis Co., Inc.), lyophilized at less than 30 itm of Hg for 6 to 8 h, and sealed under vacuum by fusing the glass with a blowtorch. The sealed vacuoles were stored at 25 to 27°C in the dark. Vacuoles containing each organism in each lyophilization medium were opened at three times after lyophilization: at 1 day, at 3 weeks, and at 3 months. Each lyophilized suspension was diluted fivefold in 2 ml of Jansen liquid medium, then incubated at 26°C and 1,280 lux. After 2 days each culture was added to 10 ml of Jansen liquid medium and incubated as before. Viability was determined by macroscopic and microscopic observation for growth of the cyanobacteria; growth was detected by the appearance of increased green color and of cells morphologically identical to those of the lyophilized organism.
777
778
APPL. ENVIRON. MICROBIOL.
CORBETT AND PARKER
RESULTS Viability after lyophilization was examined for cyanobacteria suspended in each ofthe eight media listed in Table 2. Ampules containing each organism in each suspending medium were opened at 1 day, at 3 weeks and at 3 months after lyophilization, then observed for cyanobacterial growth during several weeks of incubation. The length of the storage period between lyophilization and culturing did not detectably affect the observed viability, as is illustrated in Table 3, which gives data for samples lyophilized in lamb serum. The survival of samples lyophilized in other media was similarly unrelated to storage time. For this reason, the data from all storage times are combined in Table 2. Lyophilization of 13 cyanobacterial strains was initially attempted in egg albumin medium (Difco), in skim milk medium (Difco), and in Jansen liquid medium. Throughout 4 weeks of observation no viability was detected in any sample (Table 2), except for one ampule containing Nostoc muscorum lyophilized in Jansen medium (Table 2). Thus, none of these three media, appeared to be a satisfactory suspending agent for lyophilization of cyanobacteria. Lyophilization of the same organisms was next performed in human ascites fluid and in four different sera: lamb serum, horse serum, beef serum, and fetal bovine serum. All five of these suspending substances allowed survival of certain cultures during lyophilization (Table 2). Lamb serum protected all of the species examined and, for most organisms, provided a higher percentage of viable samples than did other suspending substances (Table 2). With the exception of Oscillatoria subbrevis, for which only one third of the lamb serum samples showed viability, at least half of the lamb serum samples of each organism survived lyophilization (Table 2). In all cases in which growth was observed, increased turbidity and increased green color were apparent after 1 week of incubation. Cultures showing growth were also observed micorscopically, revealing an increased concentration of cells morphologically identical to the cells that had been lyophilized. DISCUSSION The suspension of cyanobacteria in serum allows successful lyophilization of all organisms here examined, of which N. muscorum is the only species reported to survive lyophilization in any previously examined medium (4). Lamb serum gives the best protection of the
largest number of species. Other sera are not as effective, with both beef and fetal bovine serum yielding especially low survival frequencies. The different efficacies of the various sera could reflect differences either in protective action or in toxicity, since numerous organic compounds can inhibit the growth of cyanobacteria. Furthermore, none of the sera were treated to remove antibodies or other host resistance factors. Removal of such factors or the use of welldefined serum fractions might enhance the protective properties of serum. Interestingly, Watanabe has reported lower viability of Tolypothrix tenuis and ofCalothrix brevissima after lyophilization with bovine serum albumin than with human serum albumin (10). Many of the organisms that survived lyophilization in lamb serum have been reported not to survive lyophilization in various salts media. Thus, several species of Anacystis and ofMicrocystis (or Diplocystis) were nonviable after lyophilization both in GO salts medium (4) and in Jansen medium (Table 2). With the exception of Gloeocapsa alpicola and Gloeocapsa echinulata lyophilized in GO medium (4), nonfilamentous and nonsporulating cyanobacteria have shown no detectable viability after lyophilization in either GO (4) or Jansen medium (Table 2). Among the filamentous cyanobacteria, N. muscorum and Anabaena cylindrica retained viability after lyophilization in GO medium (4). In contrast, only N. mucorum, but not AnaTABLE 1. List of cultures Organism
uw-o culture collection no.a AblCl Ab2Cl AclCl GI Mel Ji M2
Source of culture (reference)
Axenicb
cultures
LWc Anabaena flos-aquae IU 1444 (9)e Anabaena variabilisd IU 625 (9) Anacystis nidulans LW Gloeocapsa calcarea LW Merismopedia elegans LW Microcystis aeruginosa UC 7005 (8)' Microcystis aeruginosa M4 EPA9 Microcystis aeruginosa LW NlCl Nostoc muscorum LW Osl Oscillatoria subbrevis LW Synechococcus cedrorum J3Cl IU 1191 (9) Synechococcus cedrorum SiCi IU 563 (9) Synechococcus elongatus S2C1 a UW-O, University of Wisconsin at Oshkosh. b Axenic cultures (+) were freed of bacteria by L. Corbett. -, Nonaxenic cultures. c Isolated from Lake Winnebago (LW), Wis., by G. P. Jansen. d Originally classified as Anabaena flos-aquae. e Obtained from the United States Environmental Pro'UC, University of California Culture Collection. a Obtained from the United States Environmental Protection Agency (EPA), Corvallis, Ore.
VOL. 32, 1977
LYOPHILIZED CYANOBACTERIA VIABILITY
779
TABLE 2. Viability of lyophilized cyanobacteriaa No. of vials showing viability/no. examined Organism
Egg albumin
Skim milk
me-
me-
Jansen Janen
Fetal
Human Huancte fluid
Lamb serum
Horse serum
Beef serum
bvn
serum
diumb 0/6
dium
dium du
0/6
0/6
0/3
8/9
0/6
0/3
0/6
Synechococcus cedrorum J3C1
NTC
0/3
0/3
0/3
5/6
NT
0/3
0/3
Synechococcus elongatus S2C1
0/3
NT
NT
3/3
2/5
0/3
3/5
0/3
Anacystis nidulans AclCl
NT
0/3
0/3
3/3
4/5
2/3
1/2
0/3
Microcystis aeruginosa Jl
0/4
0/2
0/2
NT
2/4
NT
NT
2/2
Microcystis aeruginosa M2
0/2
0/2
0/2
NT
2/4
NT
NT
0/2
Microcystis aeruginosa M4
0/2
0/2
0/2
NT
3/4
NT
NT
0/2
Merismopedia ekgans Mel
0/2
0/2
0/2
2/3
2/5
NT
NT
NT
Gloeocapsa calcarea Gi
0/2
0/2
0/2
NT
1/2
NT
NT
NT
Oscillatoria subbrevis Osl
0/2
0/2
0/2
0/3
1/3
NT
NT
NT
Anabaena flos-aquae AblCld
0/2
0/2
0/2
3/3
5/5
NT
2/2
NT
Anabaena variabilis Ab2Cld
0/2
0/2
0/2
NT
2/2
NT
NT
NT
2/3 Nostoc muscorum NlCld 0/2 0/2 1/2 a Viability was determined as described in Materials and Methods. b Suspending medium. c NT, Not tested. d Does form akinetes.
2/5
NT
2/5
2/2
Synechoccus cedrorum SlCl
baena flos-aquae or Anabaena variabilis, survived the somewhat different lyophilization procedures in Jansen medium (Table 2). The relative tolerance of Nostoc and Anabaena to freeze drying may arise from their production of akinetes (spores) or from other peculiarities of their structure or growth. The production of heterocysts probably did not affect their survival, since heterocysts are not believed to be formed at the high nitrogen concentrations of GO or Jansen medium and since heterocysts are not observed in Jansen medium cultures. Although species of Nostoc and Anabaena tolerate some freeze drying in salts media, the data in Table 2 indicate that the addition of lamb serum enhances the viability after lyophilization of even these organisms. Since it has been reported that skim milk solids might give some protection to certain cyanobacteria during lyophilization (4), freeze drying was attempted in skim milk medium (Difco) (Table 2). Under the conditions used here, however, skim milk did not detectably increase the viability of lyophilized cyanobac-
li
teria (Table 2). Of all organisms examined, 0. subbrevis showed the smallest fraction of viable samples, even when lyophilized in lamb serum. Although this low frequency of successful lyophilization may reflect random sampling error in a small number of samples, it is reminiscent of Holm-Hansen's observations that Oscillatoria is especially sensitive to freezing (3). In fact, resistance of cyanobacteria to freezing appears to correlate with' ease of their lyophilization, in all cases where comparisons can be made. For example, N. muscorum, which was reported to survive at least nine repeated cycles of freezing and thawing (3), can be lyophilized even in certain salts media. Synechococcus cedrorum, which was reported to survive only one freezethawing cycle (3), requires lamb serum to survive lyophilization. Oscillatoria, which did not survive even one freeze-thaw cycle (3), shows marginal survival after lyophilization, even when suspended in lamb serum. Because some cyanobacteria do not grow well at low cell densities in liquid cultures, it is
780
APPL. ENVIRON. MICROBIOL.
CORBETT AND PARKER TABLE 3. Viability after storage of cultures lyophilized in lamb seruma No. of vials showing viability/no. examined
Organism 3 3 1a"Weeks Months 1 Dayb
Anabaena flos-aquae AblCl Anabaena variabilis Ab2C2 Anacystis nidulans AclCl Gloeocapsa calcarea Gi
1/1
3/3
1/1
2/2
1/1
0/1
3/3 1/2 2/3
1/1 0/1
1/3 2/3
1/1
2/3
0/1 0/1 1/1
1/3 1/1 3/4
1/1 0/1 1/1
3/3
2/3
3/3
Synechococcus elogatus 0/1 2/3 S2C1 a Lyophilized at 26°C in the dark. b Time stored.
0/1
Merismopedia elegans Mel Microcystis aeruginosa Jl Microcystis aeruginosa M2 Microcystis aeruginosa M4 Nostoc muscorum NlCl Oscillatoria subbrevis Osl Synechococcus cedrorum J3C1 Synechococcus cedrorum
sicl
0/1
bility after 1 month of observation. The purpose of the current investigation was to develop lyophilization procedures yielding rapid and reliable growth upon cultivation of lyophilized samples, which can occur only if the number of viable cells exceeds that required for resumption of growth under standard cultivation conditions. For this reason, no attempt was made to detect small numbers of viable cells or to quantitate the fraction of surviving cells.
0/1
possible that a few viable cells may have existed in certain of the lyophilized samples, but that too few viable cells were present to allow resumption of growth and thus detection of via-
LITERATURE CITED 1. Daily, W. A., and J. M. McGuire. 1954. Preservation of some algal cultures by lyophilization. Butler Univ. Bot. Stud. 11:139-143. 2. Greaves, R. I. N. 1960. Preservation of living cells by freeze-drying. Ann. N.Y. Acad. Sci. 85:723-728. 3. Holm-Hansen, 0. 1963. Viability of blue-green and green algae after freezing. Physiol. Plant. 16:530-540. 4. Holm-Hansen, 0. 1964. Viability of lyophilized algae. Can. J. Bot. 42:127-137. 5. Holm-Hansen, 0. 1967. Factors affecting viability of lyophilized algae. Cryobiology 4:17-23. 6. Meryman, H. T. 1960. Freezing and drying of biological materials. Ann. N.Y. Acad. Sci. 85:501-734. 7. Parkes, A. S., and A. U. Smith. 1960. Recent research in freezing and drying. Blackwell Scientific Publications. Oxford. 8. Stanier, R. Y., R. Kunizawa, M., Mandel, and G. Cohen-Bazire. 1971. Purification and properties of
unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. 35:171-205. 9. Starr, R. C. 1964. The culture collection of algae at Indiana University. Am. J. Bot. 51:1013-1044. 10. Watanabe, A. 1959. Some devices for preserving bluegreen algae in viable state. J. Gen. Appl. Microbiol. 5:153-157.
Vol. 32, No. 6 Printed in U.S.A.
Viability of Lyophilized Cyanobacteria (Blue-Green Algae) LINDA L. CORBETT AND DOROTHY L. PARKER*
Biology Department, University of Wisconsin at Oshkosh, Oshkosh, Wisconsin 54901
Received for publication 11 June 1976
Lyophilization of 13 cyanobacterial cultures belonging to seven genera was attempted in a variety of suspending susbstances. All organisms survived lyophilization when suspended in lamb serum. Some of the organisms could be successfully lyophilized in horse serum, beef serum, fetal bovine serum, or human ascites fluid. With the exception of Nostoc muscorum, none of the organisms survived lyophilization in skim milk medium, egg albumin medium, or Jansen salts medium. mg; Tricine buffer (Sigma Chemical Co.), 250 mg; and sufficient 1 N NaOH to adjust the medium to pH 8.3 at 25°C. Jansen agar medium is Jansen liquid medium supplemented with 10 g of purified
Preservation of stock bacterial cultures by lyophilization is a convenient, reliable, and inexpensive procedure allowing compact storage of stock cultures (2, 6, 7). Lyophilization minimizes the hazards of genetic drift, contamination, culture death, and accidental mislabeling which can occur with routine passage. For these reasons, lyophilization is frequently the method of choice for long-term maintenance of stock cultures, when the organisms can be shown to survive the lyophilization procedure itself (2, 6, 7). Although successful lyophilization of a few species of cyanobacteria has been reported (1, 4, 5, 10), attempts to lyophilize many other cyanobacteria have failed (4, 5). Most of the cyanobacteria previously shown to survive lyophilization are filamentous organisms producing heterocysts or akinetes (spores), specialized cells which may be unusually resistant to freezing and drying. In addition, many of the lyophilizable cyanobacteria were isolated from the Antarctic (4), where frequent natural freezing and wind-produced drying may mimic lyophilization. In the present study, we have attempted to develop a lyophilization procedure that would preserve a wider variety of cyanobacteria, including several nonfilamentous and nonsporulating species isolated from the north temperate climatic zone.
agar/liter (Difco Laboratories). Suspending media for lyophilization were: skim milk medium (Difco), egg yolk enrichment medium (Difco), Jansen liquid medium, tissue culture select fetal bovine serum (Berkeley Biological Laboratories) lamb serum (Grand Island Biological Co.), beef serum (Difco), horse serum from fresh defibrinated horse blood (Grand Island Biological Co.), and human ascites fluid (Berkeley Biological Laborato-
MATERIALS AND METHODS The cyanobacteria, their origins and strain reference numbers are listed in Table 1. Jansen liquid medium contains per liter: NaNO3, 670 mg; MgSO4 -7H0, 100 mg; Na2HPO4 3H20, 17 mg; K2HPO4- 3H20, 50 mg; Na2SiO3- 5H20, 75 mg; MgCl2 6H20, 50 mg; FeCl3* 6H20, 5 mg; CaCl12 2H20, 42 mg; H3BO3, 3.25 mg; MnSO4 *4H2O, 2.4 mg; ZnSO4 7H20, 0.3 mg; (NH4)Mo71024 4H20, 0.08 mg; CuSO4-5H2O, 0.13 mg; Co(NO3)2-6H2O, 0.2 mg; KI, 0.09 mg; disodium dinitrilotetraacetate (EDTA), 4
ries). All cultures, whether in liquid or solid media, were grown without shaking at 27°C and at 1,280 lux of illumination from Westinghouse F 40-CW fluorescent lights. Before lyophilization, cultures of each organism were propagated for 10 days on the surface of Jansen agar medium in petri plates (100 by 15 mm). These cells were suspended to concentrations between 2 x 107 and 3 x 107 cells/ml by washing the agar surface with the appropriate lyophilization medium while scraping the surface with a sterile inoculation loop. A 0.5-ml portion of each cell suspension was transferred to each of several 1-ml vacuoles (Wheaton Scientific Co.), then shell frozen in propanol-dry ice. The frozen vacuoles were connected to a Virtis model 10-OlOBA lyophilizer (The Virtis Co., Inc.), lyophilized at less than 30 itm of Hg for 6 to 8 h, and sealed under vacuum by fusing the glass with a blowtorch. The sealed vacuoles were stored at 25 to 27°C in the dark. Vacuoles containing each organism in each lyophilization medium were opened at three times after lyophilization: at 1 day, at 3 weeks, and at 3 months. Each lyophilized suspension was diluted fivefold in 2 ml of Jansen liquid medium, then incubated at 26°C and 1,280 lux. After 2 days each culture was added to 10 ml of Jansen liquid medium and incubated as before. Viability was determined by macroscopic and microscopic observation for growth of the cyanobacteria; growth was detected by the appearance of increased green color and of cells morphologically identical to those of the lyophilized organism.
777
778
APPL. ENVIRON. MICROBIOL.
CORBETT AND PARKER
RESULTS Viability after lyophilization was examined for cyanobacteria suspended in each ofthe eight media listed in Table 2. Ampules containing each organism in each suspending medium were opened at 1 day, at 3 weeks and at 3 months after lyophilization, then observed for cyanobacterial growth during several weeks of incubation. The length of the storage period between lyophilization and culturing did not detectably affect the observed viability, as is illustrated in Table 3, which gives data for samples lyophilized in lamb serum. The survival of samples lyophilized in other media was similarly unrelated to storage time. For this reason, the data from all storage times are combined in Table 2. Lyophilization of 13 cyanobacterial strains was initially attempted in egg albumin medium (Difco), in skim milk medium (Difco), and in Jansen liquid medium. Throughout 4 weeks of observation no viability was detected in any sample (Table 2), except for one ampule containing Nostoc muscorum lyophilized in Jansen medium (Table 2). Thus, none of these three media, appeared to be a satisfactory suspending agent for lyophilization of cyanobacteria. Lyophilization of the same organisms was next performed in human ascites fluid and in four different sera: lamb serum, horse serum, beef serum, and fetal bovine serum. All five of these suspending substances allowed survival of certain cultures during lyophilization (Table 2). Lamb serum protected all of the species examined and, for most organisms, provided a higher percentage of viable samples than did other suspending substances (Table 2). With the exception of Oscillatoria subbrevis, for which only one third of the lamb serum samples showed viability, at least half of the lamb serum samples of each organism survived lyophilization (Table 2). In all cases in which growth was observed, increased turbidity and increased green color were apparent after 1 week of incubation. Cultures showing growth were also observed micorscopically, revealing an increased concentration of cells morphologically identical to the cells that had been lyophilized. DISCUSSION The suspension of cyanobacteria in serum allows successful lyophilization of all organisms here examined, of which N. muscorum is the only species reported to survive lyophilization in any previously examined medium (4). Lamb serum gives the best protection of the
largest number of species. Other sera are not as effective, with both beef and fetal bovine serum yielding especially low survival frequencies. The different efficacies of the various sera could reflect differences either in protective action or in toxicity, since numerous organic compounds can inhibit the growth of cyanobacteria. Furthermore, none of the sera were treated to remove antibodies or other host resistance factors. Removal of such factors or the use of welldefined serum fractions might enhance the protective properties of serum. Interestingly, Watanabe has reported lower viability of Tolypothrix tenuis and ofCalothrix brevissima after lyophilization with bovine serum albumin than with human serum albumin (10). Many of the organisms that survived lyophilization in lamb serum have been reported not to survive lyophilization in various salts media. Thus, several species of Anacystis and ofMicrocystis (or Diplocystis) were nonviable after lyophilization both in GO salts medium (4) and in Jansen medium (Table 2). With the exception of Gloeocapsa alpicola and Gloeocapsa echinulata lyophilized in GO medium (4), nonfilamentous and nonsporulating cyanobacteria have shown no detectable viability after lyophilization in either GO (4) or Jansen medium (Table 2). Among the filamentous cyanobacteria, N. muscorum and Anabaena cylindrica retained viability after lyophilization in GO medium (4). In contrast, only N. mucorum, but not AnaTABLE 1. List of cultures Organism
uw-o culture collection no.a AblCl Ab2Cl AclCl GI Mel Ji M2
Source of culture (reference)
Axenicb
cultures
LWc Anabaena flos-aquae IU 1444 (9)e Anabaena variabilisd IU 625 (9) Anacystis nidulans LW Gloeocapsa calcarea LW Merismopedia elegans LW Microcystis aeruginosa UC 7005 (8)' Microcystis aeruginosa M4 EPA9 Microcystis aeruginosa LW NlCl Nostoc muscorum LW Osl Oscillatoria subbrevis LW Synechococcus cedrorum J3Cl IU 1191 (9) Synechococcus cedrorum SiCi IU 563 (9) Synechococcus elongatus S2C1 a UW-O, University of Wisconsin at Oshkosh. b Axenic cultures (+) were freed of bacteria by L. Corbett. -, Nonaxenic cultures. c Isolated from Lake Winnebago (LW), Wis., by G. P. Jansen. d Originally classified as Anabaena flos-aquae. e Obtained from the United States Environmental Pro'UC, University of California Culture Collection. a Obtained from the United States Environmental Protection Agency (EPA), Corvallis, Ore.
VOL. 32, 1977
LYOPHILIZED CYANOBACTERIA VIABILITY
779
TABLE 2. Viability of lyophilized cyanobacteriaa No. of vials showing viability/no. examined Organism
Egg albumin
Skim milk
me-
me-
Jansen Janen
Fetal
Human Huancte fluid
Lamb serum
Horse serum
Beef serum
bvn
serum
diumb 0/6
dium
dium du
0/6
0/6
0/3
8/9
0/6
0/3
0/6
Synechococcus cedrorum J3C1
NTC
0/3
0/3
0/3
5/6
NT
0/3
0/3
Synechococcus elongatus S2C1
0/3
NT
NT
3/3
2/5
0/3
3/5
0/3
Anacystis nidulans AclCl
NT
0/3
0/3
3/3
4/5
2/3
1/2
0/3
Microcystis aeruginosa Jl
0/4
0/2
0/2
NT
2/4
NT
NT
2/2
Microcystis aeruginosa M2
0/2
0/2
0/2
NT
2/4
NT
NT
0/2
Microcystis aeruginosa M4
0/2
0/2
0/2
NT
3/4
NT
NT
0/2
Merismopedia ekgans Mel
0/2
0/2
0/2
2/3
2/5
NT
NT
NT
Gloeocapsa calcarea Gi
0/2
0/2
0/2
NT
1/2
NT
NT
NT
Oscillatoria subbrevis Osl
0/2
0/2
0/2
0/3
1/3
NT
NT
NT
Anabaena flos-aquae AblCld
0/2
0/2
0/2
3/3
5/5
NT
2/2
NT
Anabaena variabilis Ab2Cld
0/2
0/2
0/2
NT
2/2
NT
NT
NT
2/3 Nostoc muscorum NlCld 0/2 0/2 1/2 a Viability was determined as described in Materials and Methods. b Suspending medium. c NT, Not tested. d Does form akinetes.
2/5
NT
2/5
2/2
Synechoccus cedrorum SlCl
baena flos-aquae or Anabaena variabilis, survived the somewhat different lyophilization procedures in Jansen medium (Table 2). The relative tolerance of Nostoc and Anabaena to freeze drying may arise from their production of akinetes (spores) or from other peculiarities of their structure or growth. The production of heterocysts probably did not affect their survival, since heterocysts are not believed to be formed at the high nitrogen concentrations of GO or Jansen medium and since heterocysts are not observed in Jansen medium cultures. Although species of Nostoc and Anabaena tolerate some freeze drying in salts media, the data in Table 2 indicate that the addition of lamb serum enhances the viability after lyophilization of even these organisms. Since it has been reported that skim milk solids might give some protection to certain cyanobacteria during lyophilization (4), freeze drying was attempted in skim milk medium (Difco) (Table 2). Under the conditions used here, however, skim milk did not detectably increase the viability of lyophilized cyanobac-
li
teria (Table 2). Of all organisms examined, 0. subbrevis showed the smallest fraction of viable samples, even when lyophilized in lamb serum. Although this low frequency of successful lyophilization may reflect random sampling error in a small number of samples, it is reminiscent of Holm-Hansen's observations that Oscillatoria is especially sensitive to freezing (3). In fact, resistance of cyanobacteria to freezing appears to correlate with' ease of their lyophilization, in all cases where comparisons can be made. For example, N. muscorum, which was reported to survive at least nine repeated cycles of freezing and thawing (3), can be lyophilized even in certain salts media. Synechococcus cedrorum, which was reported to survive only one freezethawing cycle (3), requires lamb serum to survive lyophilization. Oscillatoria, which did not survive even one freeze-thaw cycle (3), shows marginal survival after lyophilization, even when suspended in lamb serum. Because some cyanobacteria do not grow well at low cell densities in liquid cultures, it is
780
APPL. ENVIRON. MICROBIOL.
CORBETT AND PARKER TABLE 3. Viability after storage of cultures lyophilized in lamb seruma No. of vials showing viability/no. examined
Organism 3 3 1a"Weeks Months 1 Dayb
Anabaena flos-aquae AblCl Anabaena variabilis Ab2C2 Anacystis nidulans AclCl Gloeocapsa calcarea Gi
1/1
3/3
1/1
2/2
1/1
0/1
3/3 1/2 2/3
1/1 0/1
1/3 2/3
1/1
2/3
0/1 0/1 1/1
1/3 1/1 3/4
1/1 0/1 1/1
3/3
2/3
3/3
Synechococcus elogatus 0/1 2/3 S2C1 a Lyophilized at 26°C in the dark. b Time stored.
0/1
Merismopedia elegans Mel Microcystis aeruginosa Jl Microcystis aeruginosa M2 Microcystis aeruginosa M4 Nostoc muscorum NlCl Oscillatoria subbrevis Osl Synechococcus cedrorum J3C1 Synechococcus cedrorum
sicl
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bility after 1 month of observation. The purpose of the current investigation was to develop lyophilization procedures yielding rapid and reliable growth upon cultivation of lyophilized samples, which can occur only if the number of viable cells exceeds that required for resumption of growth under standard cultivation conditions. For this reason, no attempt was made to detect small numbers of viable cells or to quantitate the fraction of surviving cells.
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possible that a few viable cells may have existed in certain of the lyophilized samples, but that too few viable cells were present to allow resumption of growth and thus detection of via-
LITERATURE CITED 1. Daily, W. A., and J. M. McGuire. 1954. Preservation of some algal cultures by lyophilization. Butler Univ. Bot. Stud. 11:139-143. 2. Greaves, R. I. N. 1960. Preservation of living cells by freeze-drying. Ann. N.Y. Acad. Sci. 85:723-728. 3. Holm-Hansen, 0. 1963. Viability of blue-green and green algae after freezing. Physiol. Plant. 16:530-540. 4. Holm-Hansen, 0. 1964. Viability of lyophilized algae. Can. J. Bot. 42:127-137. 5. Holm-Hansen, 0. 1967. Factors affecting viability of lyophilized algae. Cryobiology 4:17-23. 6. Meryman, H. T. 1960. Freezing and drying of biological materials. Ann. N.Y. Acad. Sci. 85:501-734. 7. Parkes, A. S., and A. U. Smith. 1960. Recent research in freezing and drying. Blackwell Scientific Publications. Oxford. 8. Stanier, R. Y., R. Kunizawa, M., Mandel, and G. Cohen-Bazire. 1971. Purification and properties of
unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. 35:171-205. 9. Starr, R. C. 1964. The culture collection of algae at Indiana University. Am. J. Bot. 51:1013-1044. 10. Watanabe, A. 1959. Some devices for preserving bluegreen algae in viable state. J. Gen. Appl. Microbiol. 5:153-157.
