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Preservation and storage of microorganisms in the gas phase of liquid nitrogen
CRYOBIOLOGY
10, 364367
(1973)
Preservation and Storage of Microorganisms in the Gas Phase of Liquid Nitrogen1 W. A. DAILY
AND C. E. HIGGENS
Eli Lilly and Company, Indianapolis, Indiana 46206
The preservation and storage of microorganisms are of considerable importance to the pharmaceutical and fermentation industries. Either permanent collections or short-term storage are necessary to maintain production strains, vegetative inocula for fermentation development studies, and assay organisms. Ideally, storage methods should provide conditions in which bacteria, streptomycetes, fungi, and algae are free from phenotypic changes. During recent years the storage of organisms in the liquid or the gas phase of liquid nitrogen appears to meet these requirements best (l-4). Our laboratory has used the gas phase of liquid nitrogen with uncontrolled rapid freezing and rapid thawing to facilitate the preservation of microorganisms. The advantages of using the gas phase are: 1, Cotton-plugged or plastic-capped glass tubes are used in place of hermetically sealed l-ml ampoules, thereby eliminating an ampoule-sealing device. 2. A large volume (up to 4 ml) can be stored in the glass tubes as compared with 1 ml in the ampoules. 3. The tubes are more conveniently stored and retrieved. 4. Laboratory personnel need no special safety equipment be1 From Proceedings of Japan-U.S.A. Conference on Freezing and Freeze-Drying, held October 1% 20, 1972, at Cacapon State Park, Berkeley Springs, West Virginia, sponsored by the Japan Society for the Promotion of Science, the National Science Foundation, and the American Type Culture Collection. 364 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
cause the hazard of exploding sealed glass ampoules is avoided. 5. A portion of a tubed suspension may be removed after rapid thawing and the remainder refrozen immediately in the gas phase for future work. By storing more inoculum in one tube, space is conserved in the liquid nitrogen storage unit. Since gas-phase freezing and storing is a relatively recent procedure (4), it was necessary to study the efficiency of various suspending agents for different microorganisms. This was implemented by a series of alternate freezings and thawings to hasten evaluation. MATERIAL
AND
METHODS
The protective agent was added to axenit agar slant cuhures or centrifuged broth cultures from which the supernatant fluid had been discarded. Centrifuged broth cultures also provided a source of concentrated inocula when necessary. One to four milliliters of the cell or mycelial suspension was pipetted into the tubes (13 x 100 mm, Kimblc Kimax). The tubes were either plugged with cotton or covered with plastic caps (Bellco Kap-Uts). The tubed suspensions at ambient temperature were frozen in the gas phase of liquid uitrogen at an uncontrolled cooling rate and stored in the gas phase of a Cryenco Biostat 50 M-X. When retrieved, they were thawed at 43” C until only a trace of ice remained. Then they were
INOCULA
STORAGE IN LIQUID TABLE
COMPARISON
NITROGEN
365
1
OF SUSPENDING
AGENTS
Suspending agent
Viable cells recovered of nonfrozen fresh suspension)
(Percent
Simharomycos
cereuisiue .lTCC
2366
Streptomycer tenebrarius
Lilly A-12253.1.6
~.
Trypt.icase-soy broth 10% Glycerol 570 DRISO 5% Na GMarnate 10% Polyvinyl pyrrolidine (PVP) Glucose-citrate 574 Lactose 10% PVP-5’); glycerol 5y0 DRISO-glucose-citrate 10% Glycerol-5?c lactose ___-. --________
transferred either to fresh media or into a buffer solution. Initially, over 50 potential compounds and combinations were screened for their cryoprotective utility. Only agents that were considered useful are reported in this paper. In the initial experiment, a yeast and a streptomycete were grown as vegetative cultures in shake flasks. Both cells and myCelia were prepared in 10 different agents which are listed in Table 1. The 10% DMSO-Tet. broth in Table 5 refers to dimethyl sulfoxide 10% (v/v) in the Tetrahymena broth of Hwang and Hudock (3). After being held at about -160° C in the gas phase for 1 to several days, suspensions were thawed and inoculated into shake flask media and incubated. After various periods of growth, the optical densities were recorded and the percentage of growth compared to that obtained with fresh inocula were determined. This procedure identified the suspending agent which contained the highest number of viable cells after freezing. In earlier experiments viable counts by plating were not used since mycelial forms have not produced reliable counts. It is difficult to select the best agent from a series of agents that gives consistently high recovery of viable cells when
97 127 93 97 93 108 100 9:3 115 120
86 103 70 95 67 95 68 97 100 11:s
frozen only once. We believe, however, that viable cell counts after repetitive freeze-thawings can be a most useful criterion for selecting an agent. In Tables 2 and 3, the freezings and thawings were done with less than a lomin time lapse between cycles. In Tables 4 and 5, the algal suspensions were in the frozen state for 30 min before thawing. We did not exceed six freezings and thawings with the algae since earlier work indicated rapid decline in viable cells after such treatment.
Viable cellv ~4spcrcents of nonfrozen suspension No. of times frozen at -160” C and subsequently thawed at 4R” C 1
Saccharomyccs
cercvisine
I60
8
16
500 340
ATCC 2366 Pseurlomonas aureojaciens Lilly A 10338.6
Slieptomyces lmebrurius fragmen t,irig, nr~rrs~~onllnt,irl~ strain Lilly A 122X5.1.6
110 160
39
86 106
88
366
DAILY
AND TABLE
I’MBLE
HIGGENS 3
CELLS RECOVERED FROM 28 ALTERN.WE FREEZINC+-THAWINGS (PERCENT OF NONFROZEN CELL SUSPENSIOK)
Organism
Suspending agent Trypticase-soy
Saccharomyces cereuiseae ilTCC 2366 Pseudomonas aureojaciens Lilly A-10338.6 Streptomyces tenebrariusfragmenting, nonsporulating Lilly A-12253.1.6
broth
2.3 (Tubes broken in storage) 56”
5% I)MSOcitrate
10Fer+p-
10% Glycerol5%lactose
1.0
72
> 200
1.0
155
> 200
5.6
64*
92
strain
a Results from 20 repeated freezing-thawings. b Results from 24 repeated freezing-thawings.
The species of algae used are listed in Table 5. All but Scenedesmus quadricauda, Lilly 388, were from the Indiana University (I. U.) Collection of Algae. Conventional agar diffusion assay plates were prepared by inoculating agar with a properly diluted suspension of cells in buffer. Sensitivity discs were placed on the agar, and the plates were incubated for 2448 hr. The effectiveness of a cell suspension or inoculum was determined by the sharpness and size of each zone and the relative densities of growth in the plate. RESULTS
AND
DISCUSSION
In Table 1, 10% glycerol with 570 lactose appeared to be the best cryoprotective TABLE
4
RESULTS AFTER ONE END SIX ALTT,RN~TW THAWINGS Scenedcsrnus quadricauda
Breb., Lilly Cryoprotective
Glycerol-57. Glyceroll5% Glycerol-5% Glycerold%
(Turpin)
M47-388
agent
_~~--___
107c 10% 10% 10%
FREEZING-
maltose r&nose Na glutamate lactose
Viable cella recovered &s percent of nonfrozen fresh WSpension 1 XF
6XF
79.5 70.1 81.8 64.3
45.0 66.0 41.0 26.3
agent. Presently, it is used routinely for preserving all bacteria, streptomycetes, and fungi for short- and long-term maintenance. In Table 2, an increase in viable cells as determined by plate counts is noted with up to eight alternate freezings and thawings. A noticeable drop then occurs at 16 freezings and thawings. The increase in counts of cells after freezing can be attributed to fragmentation during the alternate freezing and thawing process. Withdrawal of aliquots from the same tube over a period of time is a distinct advantage, especially in conserving storage space. Table 3 shows the results from 28 repeated freezings and thawings. On the basis of percentage of viable cells recovered, 10% glyceroM’$ lactose solution yielded the best results with all three organisms. The most efficient agents for the storage of Scenedesmus quadricaudu, Lilly 388, are listed in Table 4. A very high recovery was observed after only one freezing and thawing; 79.5-81.8$%. Another experiment with the first-listed agent (glycerol and maltose) provided 69% recovery after one freezing and thawing. The viable cell counts after six freeze-thawings with glycerol and raffinose are noteworthy. In Table 5, the glycerol-raffinose mixture
INOCULA
STORAGE IN LIQUID
NITROGEN
s. yuMlri,:mdo
$.
I.-:388
___-~~
105; Glycerol-504 maltose 10%
Glycerol-5%
r&nose
1O70Glycerol-37; Na glutamate 57, Glycerol-2.57; lactose 10% DMSO tet-broth
zones of antibiotic inhibition a 24- to 4%hr incubation.
The gas-phase frozen cells compared favorably to mlfrozen fresh cells with respect to overall growth characteristics and antibiotic
sensitivities.
S. quadricauda, Lilly 388, showed little change in sensitivity to furadantin (nitrofurantoin) after six alternate freezings and thawings
when
either
glycerol-raffinose
or
glycerol-maltose were the cryoprotective No noticeable morphological agents. changes were seen in cells or coenobia.
43.0
1.4
66.0 41.9 44.4
160.0 46.0 9.8
12.4
nidulann
I.U. 625
8.4 34.8 34.2 2.G 7.1
1.7 0.3 1.8
1.0 G.4
freezing in tubes were
the nitrogen gas phase, the stored horizontally either in
test-tube racks or in bulk containers. Frozen suspensions of vegetative cells of some algae, many genera of bacteria, as well as spores, vegetative cells, and fragmented mycclia of streptomycetes and fungi have been conveniently prepared from either agar or broth cultures. Sufficient inocula have been prepared to be
used for periods of a few weeks to several years. ACKNOWLEDGMENTS The technical assistance of Mrs. Barbara Martin Driver and Norman L. Tirmenstein, Jr., is gratcfully acknowledged. REFERENCES
Methods are described which permit the rapid preparation and rctricval of suspenstored
in the gas
space of a liquid nitrogen refrigerator. Inocula are currently used for assay plates, turbidimetric assays, bioautograph plates, and various fermentation processes. The most suitable suspending agents, 10%
glycerol with 5% of either lactose, maltose, or raffinose, were devised by determining viabilities from alternate freezing and thawing
.I nmy*tis
Four milliliters each of concentrated cell suspensions were pipetted into cottonplugged or plastic-capped glass tubes (13 x 100 mm). After rapid noncontrolled
SUMMARY
sions of microorganisms
Cf. uulgoris I.U. 30
___.~-
4,5.3
appears to be the best protective agent for the three green algae. The 160% viability recovery with Scenedemus basiliensis, I. U. 79, may be attributed to coenobial disruption. Anncystic nidulans, I. U. 625, a bluegreen alga, had a low viable cell recovery after one and six freeze-thawings. HOWever, the assay plates inoculated with this alga, after one freeze-thaw cycle, provided measurable after only
hu.~ilienais 1.1.. Til
3G7
of cell suspensions.
1. Hwang, S., and Horneland, W. Survival of algal cultures after freezing by controlled and uncontrolled cooling. Cryobiology 1, 305-311 (1965). 2. Hwang, S. Long-term preservation of fungus cultures with liquid nitrogen refrigeration. Appl. MicrobioZ. 14, 784-788 (1966). 3. Hwang, S., and Hudock, G. A. Stability of Chlamydomonas reinhardi in liquid nitrogen storage. J. Phycol. 7, 300-303 (1971). 4. McDaniel, L. E., and Bailey, E. G. Liquid nitrogen preservation of standard inoculum: gas-phase storage. AppZ. MicrobioZ. 16, 912-
916 (1968).
10, 364367
(1973)
Preservation and Storage of Microorganisms in the Gas Phase of Liquid Nitrogen1 W. A. DAILY
AND C. E. HIGGENS
Eli Lilly and Company, Indianapolis, Indiana 46206
The preservation and storage of microorganisms are of considerable importance to the pharmaceutical and fermentation industries. Either permanent collections or short-term storage are necessary to maintain production strains, vegetative inocula for fermentation development studies, and assay organisms. Ideally, storage methods should provide conditions in which bacteria, streptomycetes, fungi, and algae are free from phenotypic changes. During recent years the storage of organisms in the liquid or the gas phase of liquid nitrogen appears to meet these requirements best (l-4). Our laboratory has used the gas phase of liquid nitrogen with uncontrolled rapid freezing and rapid thawing to facilitate the preservation of microorganisms. The advantages of using the gas phase are: 1, Cotton-plugged or plastic-capped glass tubes are used in place of hermetically sealed l-ml ampoules, thereby eliminating an ampoule-sealing device. 2. A large volume (up to 4 ml) can be stored in the glass tubes as compared with 1 ml in the ampoules. 3. The tubes are more conveniently stored and retrieved. 4. Laboratory personnel need no special safety equipment be1 From Proceedings of Japan-U.S.A. Conference on Freezing and Freeze-Drying, held October 1% 20, 1972, at Cacapon State Park, Berkeley Springs, West Virginia, sponsored by the Japan Society for the Promotion of Science, the National Science Foundation, and the American Type Culture Collection. 364 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
cause the hazard of exploding sealed glass ampoules is avoided. 5. A portion of a tubed suspension may be removed after rapid thawing and the remainder refrozen immediately in the gas phase for future work. By storing more inoculum in one tube, space is conserved in the liquid nitrogen storage unit. Since gas-phase freezing and storing is a relatively recent procedure (4), it was necessary to study the efficiency of various suspending agents for different microorganisms. This was implemented by a series of alternate freezings and thawings to hasten evaluation. MATERIAL
AND
METHODS
The protective agent was added to axenit agar slant cuhures or centrifuged broth cultures from which the supernatant fluid had been discarded. Centrifuged broth cultures also provided a source of concentrated inocula when necessary. One to four milliliters of the cell or mycelial suspension was pipetted into the tubes (13 x 100 mm, Kimblc Kimax). The tubes were either plugged with cotton or covered with plastic caps (Bellco Kap-Uts). The tubed suspensions at ambient temperature were frozen in the gas phase of liquid uitrogen at an uncontrolled cooling rate and stored in the gas phase of a Cryenco Biostat 50 M-X. When retrieved, they were thawed at 43” C until only a trace of ice remained. Then they were
INOCULA
STORAGE IN LIQUID TABLE
COMPARISON
NITROGEN
365
1
OF SUSPENDING
AGENTS
Suspending agent
Viable cells recovered of nonfrozen fresh suspension)
(Percent
Simharomycos
cereuisiue .lTCC
2366
Streptomycer tenebrarius
Lilly A-12253.1.6
~.
Trypt.icase-soy broth 10% Glycerol 570 DRISO 5% Na GMarnate 10% Polyvinyl pyrrolidine (PVP) Glucose-citrate 574 Lactose 10% PVP-5’); glycerol 5y0 DRISO-glucose-citrate 10% Glycerol-5?c lactose ___-. --________
transferred either to fresh media or into a buffer solution. Initially, over 50 potential compounds and combinations were screened for their cryoprotective utility. Only agents that were considered useful are reported in this paper. In the initial experiment, a yeast and a streptomycete were grown as vegetative cultures in shake flasks. Both cells and myCelia were prepared in 10 different agents which are listed in Table 1. The 10% DMSO-Tet. broth in Table 5 refers to dimethyl sulfoxide 10% (v/v) in the Tetrahymena broth of Hwang and Hudock (3). After being held at about -160° C in the gas phase for 1 to several days, suspensions were thawed and inoculated into shake flask media and incubated. After various periods of growth, the optical densities were recorded and the percentage of growth compared to that obtained with fresh inocula were determined. This procedure identified the suspending agent which contained the highest number of viable cells after freezing. In earlier experiments viable counts by plating were not used since mycelial forms have not produced reliable counts. It is difficult to select the best agent from a series of agents that gives consistently high recovery of viable cells when
97 127 93 97 93 108 100 9:3 115 120
86 103 70 95 67 95 68 97 100 11:s
frozen only once. We believe, however, that viable cell counts after repetitive freeze-thawings can be a most useful criterion for selecting an agent. In Tables 2 and 3, the freezings and thawings were done with less than a lomin time lapse between cycles. In Tables 4 and 5, the algal suspensions were in the frozen state for 30 min before thawing. We did not exceed six freezings and thawings with the algae since earlier work indicated rapid decline in viable cells after such treatment.
Viable cellv ~4spcrcents of nonfrozen suspension No. of times frozen at -160” C and subsequently thawed at 4R” C 1
Saccharomyccs
cercvisine
I60
8
16
500 340
ATCC 2366 Pseurlomonas aureojaciens Lilly A 10338.6
Slieptomyces lmebrurius fragmen t,irig, nr~rrs~~onllnt,irl~ strain Lilly A 122X5.1.6
110 160
39
86 106
88
366
DAILY
AND TABLE
I’MBLE
HIGGENS 3
CELLS RECOVERED FROM 28 ALTERN.WE FREEZINC+-THAWINGS (PERCENT OF NONFROZEN CELL SUSPENSIOK)
Organism
Suspending agent Trypticase-soy
Saccharomyces cereuiseae ilTCC 2366 Pseudomonas aureojaciens Lilly A-10338.6 Streptomyces tenebrariusfragmenting, nonsporulating Lilly A-12253.1.6
broth
2.3 (Tubes broken in storage) 56”
5% I)MSOcitrate
10Fer+p-
10% Glycerol5%lactose
1.0
72
> 200
1.0
155
> 200
5.6
64*
92
strain
a Results from 20 repeated freezing-thawings. b Results from 24 repeated freezing-thawings.
The species of algae used are listed in Table 5. All but Scenedesmus quadricauda, Lilly 388, were from the Indiana University (I. U.) Collection of Algae. Conventional agar diffusion assay plates were prepared by inoculating agar with a properly diluted suspension of cells in buffer. Sensitivity discs were placed on the agar, and the plates were incubated for 2448 hr. The effectiveness of a cell suspension or inoculum was determined by the sharpness and size of each zone and the relative densities of growth in the plate. RESULTS
AND
DISCUSSION
In Table 1, 10% glycerol with 570 lactose appeared to be the best cryoprotective TABLE
4
RESULTS AFTER ONE END SIX ALTT,RN~TW THAWINGS Scenedcsrnus quadricauda
Breb., Lilly Cryoprotective
Glycerol-57. Glyceroll5% Glycerol-5% Glycerold%
(Turpin)
M47-388
agent
_~~--___
107c 10% 10% 10%
FREEZING-
maltose r&nose Na glutamate lactose
Viable cella recovered &s percent of nonfrozen fresh WSpension 1 XF
6XF
79.5 70.1 81.8 64.3
45.0 66.0 41.0 26.3
agent. Presently, it is used routinely for preserving all bacteria, streptomycetes, and fungi for short- and long-term maintenance. In Table 2, an increase in viable cells as determined by plate counts is noted with up to eight alternate freezings and thawings. A noticeable drop then occurs at 16 freezings and thawings. The increase in counts of cells after freezing can be attributed to fragmentation during the alternate freezing and thawing process. Withdrawal of aliquots from the same tube over a period of time is a distinct advantage, especially in conserving storage space. Table 3 shows the results from 28 repeated freezings and thawings. On the basis of percentage of viable cells recovered, 10% glyceroM’$ lactose solution yielded the best results with all three organisms. The most efficient agents for the storage of Scenedesmus quadricaudu, Lilly 388, are listed in Table 4. A very high recovery was observed after only one freezing and thawing; 79.5-81.8$%. Another experiment with the first-listed agent (glycerol and maltose) provided 69% recovery after one freezing and thawing. The viable cell counts after six freeze-thawings with glycerol and raffinose are noteworthy. In Table 5, the glycerol-raffinose mixture
INOCULA
STORAGE IN LIQUID
NITROGEN
s. yuMlri,:mdo
$.
I.-:388
___-~~
105; Glycerol-504 maltose 10%
Glycerol-5%
r&nose
1O70Glycerol-37; Na glutamate 57, Glycerol-2.57; lactose 10% DMSO tet-broth
zones of antibiotic inhibition a 24- to 4%hr incubation.
The gas-phase frozen cells compared favorably to mlfrozen fresh cells with respect to overall growth characteristics and antibiotic
sensitivities.
S. quadricauda, Lilly 388, showed little change in sensitivity to furadantin (nitrofurantoin) after six alternate freezings and thawings
when
either
glycerol-raffinose
or
glycerol-maltose were the cryoprotective No noticeable morphological agents. changes were seen in cells or coenobia.
43.0
1.4
66.0 41.9 44.4
160.0 46.0 9.8
12.4
nidulann
I.U. 625
8.4 34.8 34.2 2.G 7.1
1.7 0.3 1.8
1.0 G.4
freezing in tubes were
the nitrogen gas phase, the stored horizontally either in
test-tube racks or in bulk containers. Frozen suspensions of vegetative cells of some algae, many genera of bacteria, as well as spores, vegetative cells, and fragmented mycclia of streptomycetes and fungi have been conveniently prepared from either agar or broth cultures. Sufficient inocula have been prepared to be
used for periods of a few weeks to several years. ACKNOWLEDGMENTS The technical assistance of Mrs. Barbara Martin Driver and Norman L. Tirmenstein, Jr., is gratcfully acknowledged. REFERENCES
Methods are described which permit the rapid preparation and rctricval of suspenstored
in the gas
space of a liquid nitrogen refrigerator. Inocula are currently used for assay plates, turbidimetric assays, bioautograph plates, and various fermentation processes. The most suitable suspending agents, 10%
glycerol with 5% of either lactose, maltose, or raffinose, were devised by determining viabilities from alternate freezing and thawing
.I nmy*tis
Four milliliters each of concentrated cell suspensions were pipetted into cottonplugged or plastic-capped glass tubes (13 x 100 mm). After rapid noncontrolled
SUMMARY
sions of microorganisms
Cf. uulgoris I.U. 30
___.~-
4,5.3
appears to be the best protective agent for the three green algae. The 160% viability recovery with Scenedemus basiliensis, I. U. 79, may be attributed to coenobial disruption. Anncystic nidulans, I. U. 625, a bluegreen alga, had a low viable cell recovery after one and six freeze-thawings. HOWever, the assay plates inoculated with this alga, after one freeze-thaw cycle, provided measurable after only
hu.~ilienais 1.1.. Til
3G7
of cell suspensions.
1. Hwang, S., and Horneland, W. Survival of algal cultures after freezing by controlled and uncontrolled cooling. Cryobiology 1, 305-311 (1965). 2. Hwang, S. Long-term preservation of fungus cultures with liquid nitrogen refrigeration. Appl. MicrobioZ. 14, 784-788 (1966). 3. Hwang, S., and Hudock, G. A. Stability of Chlamydomonas reinhardi in liquid nitrogen storage. J. Phycol. 7, 300-303 (1971). 4. McDaniel, L. E., and Bailey, E. G. Liquid nitrogen preservation of standard inoculum: gas-phase storage. AppZ. MicrobioZ. 16, 912-
916 (1968).