CHYOUIOLOGY
14, 362-366 ( 1977)
Recovery of a Marine Dinoflagellate following and Uncontrolled Freezing FRANK P. SIMIOSE,
JR.
AND
PIERRE-MARC
Controlled
DAGGETT
1
American Type Culture Collection, 12301 Parklawn Drice, Rocktiille, Marylund 20852
Interest in the cryopreservation of protozoans has been predominantly restricted to parasitic forms (4). Preservation of freeliving protozoans is becoming increasingly important especially where genetic senility is a problem during long-term maintenance of genetic stocks (20). Dinoflagellates are becoming valuable in molecular biology, biochemistry, genetics, and ecology. Their nuclear characteristics are unique, demonstrating both procaryotic and eucaryotic properties ( 5, 6, 8, 16, 17, 21). The study of these unusual nuclear characteristics may provide some insight into the evolution of eukaryotic cells ( 16). Certain marine strains can be utilized in bioassaying seawater for ecologically important vitamins (7), and some strains arc used in assessing chemosensory responses to drugs (9). The discovery of sexual fusion and recombination in Crypthecodinium cohnii by the induction of slow swimming mutants (2) has drawn even greater attention to this particular dinoflagellate. Relevant to future research on these microorganisms is the retention of their characteristics during long-term uiiique maintenance. This is best achieved by storage at cryogenic temperatures. Some freeliving protozoans can be easily cryopreserved, and we are continually improving the method employed to include those groups less amenable to freezing. We re__-June 7, 1976. 1 To whon~ reprint requests should be addressed.
Received
Copyriqht 0 1977 by Academic I’rrss, Inc. All rights of reproduction in any form reserved.
port here a successful technique for the cryopreservation of C. cohnii, a free-living marine dinoflagellate. Chemical cryoprot&ants and the rate of cooling appear to be the most important criteria in low temperature preservation of protozoa ( 3). This study, therefore, involves the determination of a suitable cryoprotectant, and the effect of two cooling rates on the freezing and thawing of C. cohnii. MATERIALS
AN11 METHODS
Tolerunce Testing The sixteen strains of C. cohnii used in this study were maintained on ATCC medium 460 (1) at 25°C and were transferred by pipet once a week. Five of the strains were randomly selected for tolerance testing to dimethylsulfoxide (DMSO) and glycerol. The cells were incubated in 2.0 ml of medium 460 containing the cryoprotectants. Tolerance to each cryoprotectant was tested separately at concentrations of 5.0% (v/v), 7.5% (v/v), and lO.O(;/ (v/v).
Freezing In preparation for freezing the cultures were grown in loo-ml screw-capped bottles containing 25 ml of medium 460. After 7 days of growth, the cells were concentrated by centrifugation at 200g for 5 min and resuspended in a known volume of supernatant. An equal volume of medium 460 containing 15%) glycerol was added to the ccl1 suspension to obtain a final glycerol
1SSN
OOll-224C
CRYOPRESERVa4TION
concentration of 7.5% and a cell count of approximately 3.0 x 10G cells/ml. Aliquots of 0.5 ml of the cell suspension were dispensed into 2.0-ml polypropylene screwcapped ampoules (Vangard International Inc., No. 1076-01) and the cells were allowed to equilibrate at room tcmpcrature for 30 min. The ampoules were divided into two groups and frozen using two separate cooling rates. A controlled rate was attained by using a Linde BF-4-2 liquid nitrogen-cooled freezing chamber with a BF-6 controller. The ampoules were placed on the bottom of the freezing chamber at room temperature, and the chamber temperature was lowcrcd approximately 1 ‘C/min to -40’ C. Heat of fusion occurred at -5°C and lasted for approximately 2 min. Upon reaching -40°C the ampoules were removed from the chamber and plunged into a liquid nitrogen bath at -196°C for 5 min. A low temperature refrigerator was used to freeze the second group of samples. This resulted in an uncontrolled freezing rate. In this experiment the ampoulcs were placed on the bottom of the refrigerator at -55°C for 1 hr and were then phmged into a liquid nitrogen bath for 5 min. This effected an overall freezing rate approximately l”C, min ( 19). All ampoules were stored in a nitrogen refrigerator at -150°C for a minimum of 7 days.
OF A DINOFLACELLATE
363
ence of motile cells. Viability of recovered cells was established by survival through five transfers. RESULTS
Tolerance to Cryoprotectants Five strains were chosen randomly for tolerance testing to DMSO and glycerol and were graded on the basis of growth, retention of motility, or death while in the prescnce of the cryoprotectant (Table 1). Response to 5.0% DMSO was varied, and none of the strains survived in the presence of 7.5’2 or 10.0% DMSO. All of the strains demonstrated growth in 5.0% glycerol and either growth or retention of motility in 7.5% and lO.Ocjo glycerol. The choice of 7.5% glycerol was in part biased by earlier success with ATCC 30021 (R. Zeig, personal communication).
Recotiery Frozen material was thawed by agitating the ampoules in a 35°C water bath until complete thawing was indicated by the absence of ice in the samples. The material was not allowed to remain at 35°C beyond the completion of thawing. The percentage of intact cells recovered was determined by hemocytometer counts of a small portion of the thawed material. The remaining material was placed into 5.0 ml of medium 460 in a screw-capped test tube. Incubation was cnrricd out at 25”C, and observations were made daily for the pres-
Recovery from Freezing Frozen material was stored from 7 to 51 days at - 150°C. Recovery of intact cells from the controlled freeze ranged from 20 to 76% (Table 2). Immediately following thawing all cells were nonmotile and motility was observed within 3 to 4 days. Culturcs reached maximal growth 67 days following the appearance of the first motile cells, and all strains were viable through five transfers. Morphological characterization of the cells before and after cryopreservation indicntcd that no apparent
36-1
SIMIONE,
JR. AND
TABLIS 2 Percenlage of Intact Cells 11ec:overed following Controlled and IJncontrolletl Freezing Strain (BTCC No.)
30334 30333 30336 30337 30338 3OSS9 30340 30341 30342 30343 30344 30345 30346 30347 30348 30021 30021'~ ~. 0 After
Cmtrollrd fwwt:
~rrtYmtrollrd freew
00 3) 46 04 71 20 26 22 44 61 49 42 20 70 4.5 72 6X
17 9s 38 12 s4 20 40 so 2.3 4.-i 0s 12 24 r ,;I 12
6 years of storage at -150°C.
permanent physical damage was incurred on recovered cells. Flagella were not observed during the counting procedure on otherwise intact C. cohn,ii immediately following recovery. Recoveries following uncontrolled freezing ranged from 2.3 to 93.0% with a mean of 30.9’?. In this case motile cells were observed 5-7 days after recovery and cultures were again at maximum growth within 7 days after the appearance of the motile cells. All of the strains were viable through five transfers. The wide range in the perccntage of recovered cells in the second experimental group reflects the irregularity of the uncontrolled freezing rate. The mean recovery in this group is lower than that of the first group, and the appearance of motile cells is delayed. However the ability to establish viable cultures and survive subsequent transfer is not affected by the freezing rate. One strain, ATCC 30021, demonstrated a 68c/,, recovery following 6 years of storage at -150°C after preservation using a controlled freezing rate. Motile cells were first observed within 3 to 4 days. Cultures
DAGGETT
reached stationary phase by 10 days postrecovery. These results correspond to those for material frozen by the same method and stored for short periods of time (up to 2 months).
Cryoprotectants are necessary adjuvants for successful freeze-preservation of most protozoans, especially free-living forms. Choice of a cryoprotectant is in many casts based on previous success with an entirely different group of protozoans. The ability to tolerate the cryoprotectant is not usually considered. Lack of toxic effect is not necessarily an indication of a good cryoprotectant. However, since a period of equiIibration at ambient temperature seems to be a prerequisite to successful cryoprotection (3), a nontoxic cryoprotectant is desirable. Protozoans vary in their ability to tolerate chemical additives, therefore tolerance testing to several concentrations of the cryoprotectant is a practical first step to successful cryopreservation ( 14). Provasoli and Gold (15) d emonstrated that C. cohnii can utilize glycerol as a carbon source at concentrations of 0.2-0.6 2’. ATCC 30335 is a slow growing strain and may have demonstrated noticeable growth in 7.5$ glycerol had the testing been extended beyond 3 days. Chemicals structurally related to glycerol may provide improved cryoprotection. Although ethylene glycol demonstrates cryoprotectant properties ( 12), we have foulid it to be extremely toxic to some protozoans and have abandoned its routine use. During uncontrolled freezing there is a rapid drop in temperature until latent heat of fusion (19). The low recovery following uncontrolled freezing may result from rapid cooling, since this has been demonstrated to cause formation of intracellular ice crystals ( 18). Also, cooling continued to -55°C before the cells were subjected to supercooling, in contrast to -40°C during controlled freezing. Uncontrolled freezing, how-
CRYOPRESERVrZTIOS
OF A DINOFLACELLATE
ever, has been used with excellent results for some parasitic protozoans (Simione and Daggett, unpublished data) and for freeliving and pathogenic Naegleria ( 19). This is a less complicated procedure which may be applied more routinely than controlled freezing. Data supporting long-term storage of material following uncontrolled freezing are lacking. Material frozen by this method must be carefully monitored to insure against loss of viability during prolonged storage. Controlled freezing, on the other hand, has already proven to be suitable for use when C. cohnii is to be stored for long periods of time. The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Although only intact cells are counted following recovery, all cells observed intact are not necessarily viable. An alternate method of determining the percentage of viable cells recovered following freezing and thawing of protozoans is by diluting the thawed material and observing the number of cultures subsequently established. This technique has been reported successful for some protozoans (10, 14). Attempts in our laboratoq to develop an accurate, practical, dilution technique for measuring recovery following freeze-preservation have thus far been unsuccessful. Immobility immediately following recovery may be the result of some type of temporary physical damage. This has been reported for Euglena gracilis where motility may be delayed for as long as 10 days ( 11). Preliminary studies in our laboratory indicate that flagellar shearing may be the cause of the delayed motility in certain free-living flagellates following recovery from ultralow temperatures. Studies have not been undertaken to determine the mechanism of delayed motility following the freezing and thawing of C. cohnii. Slll\lhlAH’I A
free-living,
Crypthecodinium
marine
dinoflagcllate,
cohnii, was successfully
X5
preserved by controlled and uncontrolled freezing. Tolerance testing to various concentrations of dimethylsulfoxide (DMSO) and glycerol established that 7.5% glycerol was the best cryoprotectant. Controlled freezing was accomplished by using a biological freezer to obtain a l”C/min cooling rate. After storage for a minimum of 7 days at -150°C material frozen by this method demonstrated a 47.7%1 mean recovery, and cells were viable through five subcultures. Uncontrolled freezing resulted from placing the ampoules on the bottom of a low temperature refrigerator at -55°C for 1 hr. This material demonstrated a mean rccovery of 30.8’? with a much wider range. Cells were initially nonmotile following recovery, and in those recovered after UIICOI~trolled freezing motility was further delayed. One strain was viable after 6 years of storage with a 6S’k, recovery following controlled freezing. The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Dilution techniques have been used for nonmotile recovered cells, but this method has been unsuccessful in our laboratory. Delayed motility has been reported for other flagellates and work in our laboratory indicates that flagellar shearing may be the cwise ‘ . REFERENCES 1. Alexander, XI. T. 5ledia. In “American Type Culture Collection Cataloguc of Strains I” (II. Hatt, Ed.), pp. 318-368. American Type Cultlxe Collection, Rockvillc, hl;tryl;mtl, 1976. 2. Beam, C. A., a11c1 Himcs, hl. Evidence for scsual fusion and recombination in the dinoflagellate Cr~pthccotlinium (Gyro&&m) cohnii. Nature ( London) 250, 435436 (1974). 3. Dalgliesh, R. J. Theoretical and practical aspects of freezing parasitic protozoa. Au&t. I’d. J. 48, 233-239 ( 1972). -4. IX;m>ond, 1~. S. Frt~cl.e-pres~r\,;ltioll of protlwo~l. C:r!/ohiolog!/ 1, OS-102 ( 1964). 5. I>otlgc, J. I>. ChroII~osomc structure ill l)iliopl~yceac II. Cytochcmical strtdics. Arch. Jlikrobiol. Z. 48, 66-80 ( 1964).
366
SIMIONE,
JR. AND
6. Dodge, J. D. The Dinophyceae. In “The Chromosomes of the Algae” (M. B. E. Godward, Ed.), p. 96. Arnold, London, 1966. 7. Gold, K., and Baren, C. F. Growth requirements of Gyrodinium c&ii. J. Protozool. 13, 255-257 ( 1966). 8. Haapala, 0. K., and Soyer, M. 0. Absence of longitudinal differentiation of dinoflagellate (Prorocentrum micans) chromosomes. Here&as 78, 141-145 ( 1974). 9. Hauser, D. C. R., Levandowsky, M., and Glassgold, J. hl. Ultrasensitive chemosensory responses by a protozoan to epinephrine and other neurochemicals. Science 190, 285-286 (1975). 10. Heaf, D. P., and Lee, D. A viability assay for Tetrahymenu pyriformis. J. Gen. Micrabiol. 68, 249-251 (1971). 11. Hwang, S. W., and Horneland, W. Survival of algal cultures after freezing by controlled an d uncontrolled cooling. Cryobiology I, 305311 ( 1965). 12. Kahn, R. A., and Flinton, L. J. Evaluation of ethylene glycol as a cryoprotective agent for blood platelets. Cryobiology 10, 148-151 (1973). 13. Kubai, D. F., and Ris, H. Division in the dinoflagellate Gyrodinium cohnii Schiller. A new type of nuclear reproduction, J. Cell Bid. 40, 508-528 (1969). 14. Osborne, J. A., and Lee L1. Studies on the con-
15.
16.
17.
18.
19.
20.
21.
DAGGETT ditions required for optimum recovery of Tetrahymena pyriformis strain S (Phenoset A) after freezing to and thawing from -196°C. J. Protozool. 22, 233-237 (1975). Provasoli, L., and Gold, K. Nutrition of the American Strain of Gyrodinium cohnii. Arch. f MikrobioZ. Z. 42, 196-203 ( 1962). Rizzo, P. J., and Nooden, L. D. Chromosomal proteins in the dinoflagellate alga Gyrodinium cohnii. Science 176, 796-797 (1972). Roberts, T. M., Tuttle, R. C., Allen, J. R., Loeblich, A. R. III, and Klotz, L. C. New genetic and physicochemical data on structure of dinoflagellate chromosomes. Nature (London) 248, 446447 ( 1974). Shimada, K., and Asahina, E. Visualization of intracellular ice crystals formed in very rapidly frozen cells at -27°C. Cryobiology 12, 209-218 ( 1975). Simione, F. P. Jr., and Daggett, P.-M. Freeze preservation of pathogenic and nonpathogenie Naegleria species. .I. Parasit. 62, 49 (1976). Simon, E. M., and Schneller, M. V. The preservation of ciliated protozoa at low tcmperature. Cryobiology 10, 421426 ( 1973). Soyer, M. 0. Structure de noyaux de Blastodinium ( Dinoflagelles parasites). Division et condensation chromatique. Chromosoma 39, 70-114 (1971).
14, 362-366 ( 1977)
Recovery of a Marine Dinoflagellate following and Uncontrolled Freezing FRANK P. SIMIOSE,
JR.
AND
PIERRE-MARC
Controlled
DAGGETT
1
American Type Culture Collection, 12301 Parklawn Drice, Rocktiille, Marylund 20852
Interest in the cryopreservation of protozoans has been predominantly restricted to parasitic forms (4). Preservation of freeliving protozoans is becoming increasingly important especially where genetic senility is a problem during long-term maintenance of genetic stocks (20). Dinoflagellates are becoming valuable in molecular biology, biochemistry, genetics, and ecology. Their nuclear characteristics are unique, demonstrating both procaryotic and eucaryotic properties ( 5, 6, 8, 16, 17, 21). The study of these unusual nuclear characteristics may provide some insight into the evolution of eukaryotic cells ( 16). Certain marine strains can be utilized in bioassaying seawater for ecologically important vitamins (7), and some strains arc used in assessing chemosensory responses to drugs (9). The discovery of sexual fusion and recombination in Crypthecodinium cohnii by the induction of slow swimming mutants (2) has drawn even greater attention to this particular dinoflagellate. Relevant to future research on these microorganisms is the retention of their characteristics during long-term uiiique maintenance. This is best achieved by storage at cryogenic temperatures. Some freeliving protozoans can be easily cryopreserved, and we are continually improving the method employed to include those groups less amenable to freezing. We re__-June 7, 1976. 1 To whon~ reprint requests should be addressed.
Received
Copyriqht 0 1977 by Academic I’rrss, Inc. All rights of reproduction in any form reserved.
port here a successful technique for the cryopreservation of C. cohnii, a free-living marine dinoflagellate. Chemical cryoprot&ants and the rate of cooling appear to be the most important criteria in low temperature preservation of protozoa ( 3). This study, therefore, involves the determination of a suitable cryoprotectant, and the effect of two cooling rates on the freezing and thawing of C. cohnii. MATERIALS
AN11 METHODS
Tolerunce Testing The sixteen strains of C. cohnii used in this study were maintained on ATCC medium 460 (1) at 25°C and were transferred by pipet once a week. Five of the strains were randomly selected for tolerance testing to dimethylsulfoxide (DMSO) and glycerol. The cells were incubated in 2.0 ml of medium 460 containing the cryoprotectants. Tolerance to each cryoprotectant was tested separately at concentrations of 5.0% (v/v), 7.5% (v/v), and lO.O(;/ (v/v).
Freezing In preparation for freezing the cultures were grown in loo-ml screw-capped bottles containing 25 ml of medium 460. After 7 days of growth, the cells were concentrated by centrifugation at 200g for 5 min and resuspended in a known volume of supernatant. An equal volume of medium 460 containing 15%) glycerol was added to the ccl1 suspension to obtain a final glycerol
1SSN
OOll-224C
CRYOPRESERVa4TION
concentration of 7.5% and a cell count of approximately 3.0 x 10G cells/ml. Aliquots of 0.5 ml of the cell suspension were dispensed into 2.0-ml polypropylene screwcapped ampoules (Vangard International Inc., No. 1076-01) and the cells were allowed to equilibrate at room tcmpcrature for 30 min. The ampoules were divided into two groups and frozen using two separate cooling rates. A controlled rate was attained by using a Linde BF-4-2 liquid nitrogen-cooled freezing chamber with a BF-6 controller. The ampoules were placed on the bottom of the freezing chamber at room temperature, and the chamber temperature was lowcrcd approximately 1 ‘C/min to -40’ C. Heat of fusion occurred at -5°C and lasted for approximately 2 min. Upon reaching -40°C the ampoules were removed from the chamber and plunged into a liquid nitrogen bath at -196°C for 5 min. A low temperature refrigerator was used to freeze the second group of samples. This resulted in an uncontrolled freezing rate. In this experiment the ampoulcs were placed on the bottom of the refrigerator at -55°C for 1 hr and were then phmged into a liquid nitrogen bath for 5 min. This effected an overall freezing rate approximately l”C, min ( 19). All ampoules were stored in a nitrogen refrigerator at -150°C for a minimum of 7 days.
OF A DINOFLACELLATE
363
ence of motile cells. Viability of recovered cells was established by survival through five transfers. RESULTS
Tolerance to Cryoprotectants Five strains were chosen randomly for tolerance testing to DMSO and glycerol and were graded on the basis of growth, retention of motility, or death while in the prescnce of the cryoprotectant (Table 1). Response to 5.0% DMSO was varied, and none of the strains survived in the presence of 7.5’2 or 10.0% DMSO. All of the strains demonstrated growth in 5.0% glycerol and either growth or retention of motility in 7.5% and lO.Ocjo glycerol. The choice of 7.5% glycerol was in part biased by earlier success with ATCC 30021 (R. Zeig, personal communication).
Recotiery Frozen material was thawed by agitating the ampoules in a 35°C water bath until complete thawing was indicated by the absence of ice in the samples. The material was not allowed to remain at 35°C beyond the completion of thawing. The percentage of intact cells recovered was determined by hemocytometer counts of a small portion of the thawed material. The remaining material was placed into 5.0 ml of medium 460 in a screw-capped test tube. Incubation was cnrricd out at 25”C, and observations were made daily for the pres-
Recovery from Freezing Frozen material was stored from 7 to 51 days at - 150°C. Recovery of intact cells from the controlled freeze ranged from 20 to 76% (Table 2). Immediately following thawing all cells were nonmotile and motility was observed within 3 to 4 days. Culturcs reached maximal growth 67 days following the appearance of the first motile cells, and all strains were viable through five transfers. Morphological characterization of the cells before and after cryopreservation indicntcd that no apparent
36-1
SIMIONE,
JR. AND
TABLIS 2 Percenlage of Intact Cells 11ec:overed following Controlled and IJncontrolletl Freezing Strain (BTCC No.)
30334 30333 30336 30337 30338 3OSS9 30340 30341 30342 30343 30344 30345 30346 30347 30348 30021 30021'~ ~. 0 After
Cmtrollrd fwwt:
~rrtYmtrollrd freew
00 3) 46 04 71 20 26 22 44 61 49 42 20 70 4.5 72 6X
17 9s 38 12 s4 20 40 so 2.3 4.-i 0s 12 24 r ,;I 12
6 years of storage at -150°C.
permanent physical damage was incurred on recovered cells. Flagella were not observed during the counting procedure on otherwise intact C. cohn,ii immediately following recovery. Recoveries following uncontrolled freezing ranged from 2.3 to 93.0% with a mean of 30.9’?. In this case motile cells were observed 5-7 days after recovery and cultures were again at maximum growth within 7 days after the appearance of the motile cells. All of the strains were viable through five transfers. The wide range in the perccntage of recovered cells in the second experimental group reflects the irregularity of the uncontrolled freezing rate. The mean recovery in this group is lower than that of the first group, and the appearance of motile cells is delayed. However the ability to establish viable cultures and survive subsequent transfer is not affected by the freezing rate. One strain, ATCC 30021, demonstrated a 68c/,, recovery following 6 years of storage at -150°C after preservation using a controlled freezing rate. Motile cells were first observed within 3 to 4 days. Cultures
DAGGETT
reached stationary phase by 10 days postrecovery. These results correspond to those for material frozen by the same method and stored for short periods of time (up to 2 months).
Cryoprotectants are necessary adjuvants for successful freeze-preservation of most protozoans, especially free-living forms. Choice of a cryoprotectant is in many casts based on previous success with an entirely different group of protozoans. The ability to tolerate the cryoprotectant is not usually considered. Lack of toxic effect is not necessarily an indication of a good cryoprotectant. However, since a period of equiIibration at ambient temperature seems to be a prerequisite to successful cryoprotection (3), a nontoxic cryoprotectant is desirable. Protozoans vary in their ability to tolerate chemical additives, therefore tolerance testing to several concentrations of the cryoprotectant is a practical first step to successful cryopreservation ( 14). Provasoli and Gold (15) d emonstrated that C. cohnii can utilize glycerol as a carbon source at concentrations of 0.2-0.6 2’. ATCC 30335 is a slow growing strain and may have demonstrated noticeable growth in 7.5$ glycerol had the testing been extended beyond 3 days. Chemicals structurally related to glycerol may provide improved cryoprotection. Although ethylene glycol demonstrates cryoprotectant properties ( 12), we have foulid it to be extremely toxic to some protozoans and have abandoned its routine use. During uncontrolled freezing there is a rapid drop in temperature until latent heat of fusion (19). The low recovery following uncontrolled freezing may result from rapid cooling, since this has been demonstrated to cause formation of intracellular ice crystals ( 18). Also, cooling continued to -55°C before the cells were subjected to supercooling, in contrast to -40°C during controlled freezing. Uncontrolled freezing, how-
CRYOPRESERVrZTIOS
OF A DINOFLACELLATE
ever, has been used with excellent results for some parasitic protozoans (Simione and Daggett, unpublished data) and for freeliving and pathogenic Naegleria ( 19). This is a less complicated procedure which may be applied more routinely than controlled freezing. Data supporting long-term storage of material following uncontrolled freezing are lacking. Material frozen by this method must be carefully monitored to insure against loss of viability during prolonged storage. Controlled freezing, on the other hand, has already proven to be suitable for use when C. cohnii is to be stored for long periods of time. The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Although only intact cells are counted following recovery, all cells observed intact are not necessarily viable. An alternate method of determining the percentage of viable cells recovered following freezing and thawing of protozoans is by diluting the thawed material and observing the number of cultures subsequently established. This technique has been reported successful for some protozoans (10, 14). Attempts in our laboratoq to develop an accurate, practical, dilution technique for measuring recovery following freeze-preservation have thus far been unsuccessful. Immobility immediately following recovery may be the result of some type of temporary physical damage. This has been reported for Euglena gracilis where motility may be delayed for as long as 10 days ( 11). Preliminary studies in our laboratory indicate that flagellar shearing may be the cause of the delayed motility in certain free-living flagellates following recovery from ultralow temperatures. Studies have not been undertaken to determine the mechanism of delayed motility following the freezing and thawing of C. cohnii. Slll\lhlAH’I A
free-living,
Crypthecodinium
marine
dinoflagcllate,
cohnii, was successfully
X5
preserved by controlled and uncontrolled freezing. Tolerance testing to various concentrations of dimethylsulfoxide (DMSO) and glycerol established that 7.5% glycerol was the best cryoprotectant. Controlled freezing was accomplished by using a biological freezer to obtain a l”C/min cooling rate. After storage for a minimum of 7 days at -150°C material frozen by this method demonstrated a 47.7%1 mean recovery, and cells were viable through five subcultures. Uncontrolled freezing resulted from placing the ampoules on the bottom of a low temperature refrigerator at -55°C for 1 hr. This material demonstrated a mean rccovery of 30.8’? with a much wider range. Cells were initially nonmotile following recovery, and in those recovered after UIICOI~trolled freezing motility was further delayed. One strain was viable after 6 years of storage with a 6S’k, recovery following controlled freezing. The lack of motility immediately following recovery leads to inaccuracies when determining the percentage of cells recovered. Dilution techniques have been used for nonmotile recovered cells, but this method has been unsuccessful in our laboratory. Delayed motility has been reported for other flagellates and work in our laboratory indicates that flagellar shearing may be the cwise ‘ . REFERENCES 1. Alexander, XI. T. 5ledia. In “American Type Culture Collection Cataloguc of Strains I” (II. Hatt, Ed.), pp. 318-368. American Type Cultlxe Collection, Rockvillc, hl;tryl;mtl, 1976. 2. Beam, C. A., a11c1 Himcs, hl. Evidence for scsual fusion and recombination in the dinoflagellate Cr~pthccotlinium (Gyro&&m) cohnii. Nature ( London) 250, 435436 (1974). 3. Dalgliesh, R. J. Theoretical and practical aspects of freezing parasitic protozoa. Au&t. I’d. J. 48, 233-239 ( 1972). -4. IX;m>ond, 1~. S. Frt~cl.e-pres~r\,;ltioll of protlwo~l. C:r!/ohiolog!/ 1, OS-102 ( 1964). 5. I>otlgc, J. I>. ChroII~osomc structure ill l)iliopl~yceac II. Cytochcmical strtdics. Arch. Jlikrobiol. Z. 48, 66-80 ( 1964).
366
SIMIONE,
JR. AND
6. Dodge, J. D. The Dinophyceae. In “The Chromosomes of the Algae” (M. B. E. Godward, Ed.), p. 96. Arnold, London, 1966. 7. Gold, K., and Baren, C. F. Growth requirements of Gyrodinium c&ii. J. Protozool. 13, 255-257 ( 1966). 8. Haapala, 0. K., and Soyer, M. 0. Absence of longitudinal differentiation of dinoflagellate (Prorocentrum micans) chromosomes. Here&as 78, 141-145 ( 1974). 9. Hauser, D. C. R., Levandowsky, M., and Glassgold, J. hl. Ultrasensitive chemosensory responses by a protozoan to epinephrine and other neurochemicals. Science 190, 285-286 (1975). 10. Heaf, D. P., and Lee, D. A viability assay for Tetrahymenu pyriformis. J. Gen. Micrabiol. 68, 249-251 (1971). 11. Hwang, S. W., and Horneland, W. Survival of algal cultures after freezing by controlled an d uncontrolled cooling. Cryobiology I, 305311 ( 1965). 12. Kahn, R. A., and Flinton, L. J. Evaluation of ethylene glycol as a cryoprotective agent for blood platelets. Cryobiology 10, 148-151 (1973). 13. Kubai, D. F., and Ris, H. Division in the dinoflagellate Gyrodinium cohnii Schiller. A new type of nuclear reproduction, J. Cell Bid. 40, 508-528 (1969). 14. Osborne, J. A., and Lee L1. Studies on the con-
15.
16.
17.
18.
19.
20.
21.
DAGGETT ditions required for optimum recovery of Tetrahymena pyriformis strain S (Phenoset A) after freezing to and thawing from -196°C. J. Protozool. 22, 233-237 (1975). Provasoli, L., and Gold, K. Nutrition of the American Strain of Gyrodinium cohnii. Arch. f MikrobioZ. Z. 42, 196-203 ( 1962). Rizzo, P. J., and Nooden, L. D. Chromosomal proteins in the dinoflagellate alga Gyrodinium cohnii. Science 176, 796-797 (1972). Roberts, T. M., Tuttle, R. C., Allen, J. R., Loeblich, A. R. III, and Klotz, L. C. New genetic and physicochemical data on structure of dinoflagellate chromosomes. Nature (London) 248, 446447 ( 1974). Shimada, K., and Asahina, E. Visualization of intracellular ice crystals formed in very rapidly frozen cells at -27°C. Cryobiology 12, 209-218 ( 1975). Simione, F. P. Jr., and Daggett, P.-M. Freeze preservation of pathogenic and nonpathogenie Naegleria species. .I. Parasit. 62, 49 (1976). Simon, E. M., and Schneller, M. V. The preservation of ciliated protozoa at low tcmperature. Cryobiology 10, 421426 ( 1973). Soyer, M. 0. Structure de noyaux de Blastodinium ( Dinoflagelles parasites). Division et condensation chromatique. Chromosoma 39, 70-114 (1971).
