Preservation of bacteria by freezing at moderately low temperatures

Preservation of bacteria by freezing at moderately low temperatures

CRYOBIOLOGY 10, 453463 (1973) Preservation of Bacteria by Freezing at Moderately Low Temperatures ’ KAZUHIDE Institute YIIMASATO, of Applied DAIJ...

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CRYOBIOLOGY

10, 453463 (1973)

Preservation of Bacteria by Freezing at Moderately Low Temperatures ’ KAZUHIDE Institute

YIIMASATO,

of Applied

DAIJ

OKUNO,

Microbiology,

The University

There are two lines of research for developing preservation methods for microorganisms. One is to develop a specially designed method for delicate microorganisms which are difficult or impossible to preserve by already established routine methods. The second is to develop mcthods for long-term preservation which are as simple as possible and are able to cover as wide a variety of microorganisms as possible. Freeze-preservation in liquid nitrogen is an excellent method and is used mainly for organisms to which freeze-drying cannot be applied. But sometimes it is not so easy to obtain such an extremely low temperature in ordinary laboratories. On the other hand a moderately low temC or -80” C is perature such as -28” easily provided by electric refrigerators, and its maintenance is not troublesome. Since freeze-preservation minimizes genetic alteration which may be caused by drying cells ( 12, 13) and its procedures are simple and save time and labor, it is worth investigating whether it is as a means .-1 From Proceedings of Japan-U.S.A. Conference on Freezing and Freeze-Drying, held October 1520, 1972, at Cacapon State Park, Berkeley SpringT, West Virginia, sponsored by the Japan Society for the Promotion of Science, The National Science Foundation, and the American Type Culture Collection. 2 Present address: Department of Microbiology, St. Marianna University School of Medicine, Kawasaki, Japan.

of Tokyo,

OHTOhlO Tokyo,

2

Japan

applicable to a wide variety of microorganisms. The bacteria1 strains of several species h avc been reported to have been successfully preserved by freezing at moderately low temperatures. Pasteurella pestis was reported to have been we11 preserved at -23” C for 4 years by Mead et al. (S), Bordetella pertussk at -70” C for 45 months by Eckert and Flaherty (3), Escherichia coli at -40” C and -78” C for 28 months by Clement (2), and SaZmoneZZa typhosa, Salmonella cholera&s, Bacillus anthrasis, Streptococcus ptyogenes, Shigella sonnei, Vibrio comma and Klebsielln pneumoniae at -76” C for 2 years by Yurchenco et al. (14). Unsuccessful results have also been reported. The results which TABLE

1

STRAIKS, SPIXIKS, AND GRXERA OF BACTF:RIA USED IN TIIIS STUDY -__ Strain

Group of hackvia”

Genus

Species

Aerobic gram-positives Aerobic gram-neg:Ltives Psychrophilic bacteria Lactic acid bacteriab Acet,ic acid bacteria S(ored at -5::” C Stored at -28” C Total

10 13 5 5

40 35 8 22

101 72 20 39

2 2 32

20 19 135

35 27 239

-.- _..

-~

a TJsed strains were divided into five groups and referred to as such in the text. b One strain of Propionibacterium was included in the group for convenience. 453

Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

ASP TOSHICHIKA

YAMASATO,

454

OKUNO,

AND

Agar slant culture

OHTOMO Broth culture

+ Harvested by centrifugation Aerobic bacteria (lO~“‘~/ml)~

11 Washedwith physiological

Acetic acid bacteria (lO’~““/rnl)

c

Suspendedin 10%glycerol and 10%honey I

t

t

Equilibrated at 5”CforlwPhr

Equilibrated at 5°C for 2 hr

t

+ Frozen in L.N.

Stored at -53°C for 16 months

1 Stored at 28°C for 4.5 y’ ‘I Initial cell concentration before freezing. FIG. 1. The outline

of freezing

TABLE

procedures.

2

COMPOSITION OF iMe~r.4 USED FOR CELL HARVEST AND RECOVERY For viable cell counting

For cell harvest Aerobic bacteria Psychrophiles

Nurtient aga?J Polypeptone Yeast extract Glucose Dist. water

Lactic

Polypeptone Yeast extract Glucose KHSOa Na-thioglycolale Dist. water Yeast extract Beef extract Glucose Glycerol 207~ Potato ~xt~mt

acid bacteria”

Acetic acid bacteria

10 g s!z 10 g 1 liter

10 g 5g 20 g .i g lg 1 liter 30 g 5g 5g 15 g 1 liirl~

Trypticase soy agarb Polypeptone 10 g Yeast extract 5g Glucose 10 !4 1 liter Dist. water Agar Polypeptone 10 g 5g Yeast extract 10 g Glucose 2g KHgP04 Na-t,hioglycolnt,e 1g 1 liter Dist. wat,er Mannitol sgar

__--~ n Laclohaci//~~s Zcichmannii: 1,eictrnlannii Inocul:tt.ion nledium (Nissan). .\Iedia for some strains were added with several kinds of peptone (see Table 8). b For gaseous hydrocarbon-utilizing strains Pridham’s yeast extract-malt extract medium cell harvest (broth) and recovery (with agar).

was

used

for

FREEZE-PRESERVATION TABLE

3

VI,~~I~ITY OF AEROBIC GR.*M-POSITIVE BXTERLI her of ber of species strains

.Ilicrococcu,s

8

1.3

Sarcinn

7

Stnph~lococcus Bncillrrs

2

11 6 11

l~rcvibactwiurn Coryncbactwium Arthrobacter Cdlulomonas

9 4

7

14 1 4 4

:ll~cobacterium A4~ocarrlia

AFTKR FROZEN STORAW

~~ ~-~ 0 10 102 10” 10’ 10” IO” 10’

1"

1

1

3 1

2

19 1

3

2 6 2 1 2

1 1

108 108 10’0

I

1

12 16 1 4 8

455

OF BACTERIA

2 2 2 3 5 1 2 5

1

1

-5

I

1

13/13

4 1

2

1

0 7 5

4 1 3

2

s/11 :I/‘6 ll/ll 12,/17 lO/lO 12,;13 O/l / /

1

a Number of strains which gave viable cell counts between 10 and lo* per ml after storage in 107e glycerol at -53” C for 16 months. * Viability after storage at 10” C for 2 years.

conflict with each other may be as a result of the differences between strains used and methods or procedures employed by different authors. In order to get some basic information for developing a long-term preservation method at moderately low temperatures viability spectra of bacteria with respect to groups, genera, species, and strains are de-

scribed and some discussions are presented in this study. MATERIALS

A4icroorganisms. in this study were culture collection plied Microbiology, (IAM) and those

TABLE -__--

__~-~, GXIIW

10

10

2 7

2

1

17 3

2

8

Klsbsiella

1 1

1

Swxtia Proteus

Erwinia Achromobacter Alcaligencs Flavobacterium A cfin~ohnrillus

METHODS

Most of the strains used those maintained in the of the Institute of ApUniversity of Tokyo derived from other cul-

4

---

NUKIher of her of speck3 strains

Viable cells/ml

Num-

Xanthomonas Arrottzonas Eschcrichia Entcrobactet

Psr udotnonas

AND

2 6 6

2 2 8 6

1

8

2

4

1

1

0

10’ 10’ 10” 10’ 10% 106 10’

lb

1

4

108 109 10’0

2

Liquid paraffin seals Number of Number of strains strains surviving / tested

2

lO/lO

1

16/17 O/l 4!8 l/l

2 1

2

4

2 2 1

l/l 9 3 2

3 2 2

2

3 1

1

2

o/2

1

1

3 2 5 2

2

2;2 5/8 5/G 7/g 2/4

1 __ .---

~- ~___

0,/l .-__

“ Viabilit,y after storage at 10” C for 2 years. b Nurnljer of strains which gave viable ccl1 counts between 0 and 10 per ml after storage in lO0/0 glycerol -5.1” C for 16 months.

at

456

YAMASATO,

OKUNO, AND OHTOMO TABLE

5

COMP:\RISON OF VIMHLITI~;S OF GR.~M-POSITIVE AND GUM-XICGATIVE Ak~~~~ FROZEN STORAGE -IT -5X” C FOR 16 MONTHS -___---

B.\CTXRI\

-..--

-___ Total

Viable cells/ml 0

Grampositive

Gramnegative

Number of strains Proportion (70) Number of strains Proportion (o/u)

10’

102

0

2

15

0

2.0

1.0

1

0

1.4 0 ~~ .~

103

10’

106

2

4

15

2,i

32

11

4

101

5.0

2.0

4.0

14.8

24.8

31.7

10.9

4.0

100

0

1

0

5

9

26

17

12

1

72

0

1.4

0

6.9

12.5

36.1

23.6

ture collections. Some strains were obtained from H. Iizuka, N. Seto, S. Sakayanagi, and K. Inoue (Institute of Applied Microbiology, University of Tokyo). The number of tested strains, species, and genera are listed in Table 1. 32 genera, 135 species, and 259 strains were tested. These are tentatively divided into five groups as shown in the Table and referred to as such in the text. Procedures for freezing, storage, and viable cell counting. The outline of freezing procedures and the initial cell concentrations are shown in Fig. 1. The cells of aerobic bacteria were grown on agar slants and suspended in aqueous solutions of 10% glycerol and stored at -53” C for 16 months. The cells of the remaining four groups of bacteria were grown in liquid media and suspended in 10% glycerol and stored at -53” C for 16 months. In other experiments the cells of acetic acid bacteria were suspended in two aqueous solutions of 10% glycerol and 10% honey, frozen in liquid nitrogen, and stored at -28” C for 4.5 years. The composition of media used for cell harvest and recovery are described in Table 2. After storage the frozen cells were thawed in running tap water within 1 or 2 min, diluted with 0.5% peptone water, and plated on agar media. The viability of a strain was expressed as the number of cells per milliliter which survived frozen storage. The number of

106

10’

108

109 10’0 ______~

___.

16.7 1.4 -_____

100 -

viable cells of lactic acid bacteria was calculated from the maximum dilution with liquid media which gave growth. In parallel with the frozeu storage two methods of preservation were conducted; all the strains were preserved under a liquid paraffin seal at 10” C for 2 years, and the strains of lactic and acetic acid bacteria were preserved by freeze-drying. RESULTS

AND

DISCUSSION

Aerobic bacteria. The viability of the strains of aerobic, gram-positive, and gramnegative bacteria are summarized in Tables TABLE VIABILITY

6

OF PSYCHROPHILIC BACTERW FROZEN STORAGE AT -53" C FOR 16 MONTHS -__ Species

Strain no. -I_~.

Arthrobactcr sp. Micrococcus cryophilus Cytophaga sp. Cytophaga sp. Cytophaga sp. Cytophaga sp. Cytophaga sp. Pseudomonas sp. Pseudomonas p~rtrcfacicns Spirillum sp. ~~

27-O-b 72-o-c 5-O-d 13-O-d 16-O-d 85-0-a 5-o-c 22-0-d 65-O-a 27-0-d -~--.-

AFTER

Viable cells/ml

10 9 x 105 4 x 102 0 0 8 x 108 4 x 108 102 0 4 x lo”

a These strains were collected in Antarctica by K. Inoue (Institute of Applied %Iicrobiology, University of Tokyo).

FREEZE-PRESERVATION

10'

102

10,'

101

105

0112 0 2.6

2.6

3.1

3 7.7

8 20..5

0

Number of strains Proport,lon (‘To)

3 and 4, respectively. The viability of each genus is seen in the distribution of the number of strains at various levels of viable cell counts. Of these 173 strains, 9370, SS%,, and 74% of the strains gave viable cell counts higher than 105/ml, lO”/ml, and 107/ml, respectively. There stems to be some genus- or group-specificity in resistance to frozen storage. Coryneform bacteria including the genera Brevibacterium, Corynebacterium, and Arthrobacter were resistant. The genera of the family Enterobacteriaceae except for the genus Erzoinia constitute a resistant group. The strains of low viability were observed in cocci including the genera Micrococcw, Surcina, and Staphylococcus, and the genera Nocardia and Aeromonus. Pseudomonas strains were resistant, except for one strain of P. putrefaciens which did not survive. Since another strain of the species-which is an

457

OF BACTERIA

1Ob

12 30.8

10’

108

8 20.5

4

0

10.3

0

hclvcticus ddbrumkii delbrurckii l&is

StreptococccLs hclis

39 100

obligate psychrophile-was also nonviable (Table 6) and the other two mesophilic strains gave low viable cell counts by freeze-drying, this species may be considered to be sensitive to freezing treatment. Considering that P. putrefacicns is not a typical pseudomonad and will be excluded from the genus, Pseudomonas may be considered to survive frozen storage well. The viabilities of gram-positive and gram-negative bacteria are compared in Table 5. No remarkable differences were observed except in the proportion of the sensitive strains which gave cell counts lower than 105/ml. These strains were 10% for gram-positives and 370 for gram-negatives; gram-positives are seemingly more sensitive to frozen storage. Although it is worth noticing that this is contradictory to the empirical data that gram-positives are more resistant to freeze-drying, this differ-

1\Iedium

Lactobacillus Lactobacillus Lactobacillus Lactobacill~~s

109

16

X\lediurn

IAAI'"104:! 1.411 1148

102 101

I.ZlI Ii211

10’ 104

106 104 10” 10”

10'

10'

1174 I 17::

IAhI 124!)

11~

a Number of viable cells,/ml after storage in 10’10 glycerol at -53” C for 16 ~nont~hs. b Medium I : Polypeptone (Daigo), 1.0 ‘Ir,; yeast, extract, 0.57;; glu(‘osc, 1.070; KHzPOd, 0.2.jcl,; Na-thioglycolate, 0.1 ‘,th; pH 7.0. c Medium II : Brain-heart infusion (Oxoid), l.8.iC,“, ; tryptone (Oxoid), 0.37,; trypticase (BBL), 0.3%; phytone (BBL), 0.3’;‘,; ‘Lab-Lemco’ powder (oxoid), 0.37;; yeast extract, 0.3?$; glucose, 1.055; KH*PO+ 0.125%; pH 7.0. d Institute of Applied Microbiology, University of Tokyo.

458

YAMASATO,

OKUNO,

AND

TABLE

Species

“Intermediate”

9

Strain no.

Genus Acetobacter A. aceli A. acetosus A. acctosus A. acetosus A. ascendtm A. ascendens A. kuetzingianus A. pastorianus A. rancens A. rancens var. I A. sp.

OHTOMO

Viable cells/ml~

Lyophile”

IAM 1802 IAhI 1803 IAM 1805 IAM 1804 IAll 1808 IAhI 1811 IAhI 1817 IAM 1824 IAM 1825 TAM 1826 A-23

6 X lo7 3 x 108
+ +

IAbI IAM IAM

+ /

+ + + +

+ + + + +

/ + + +

/ + + +

3 x 109 3 x 107 5 x 104

+ + +

-

5 2 8 3 4

+ + + + + + + +

-

strain

G. liquefaciens G. melanogenus G. melanogenus

1834 1835 1836

Genus Gluconobacter G. albidus G. capsulatus G. cerinus G. dioxyacetonicus G. industrius G. gluconicus G. melanogenus G. melanogenus G. melanogenus G. melanogenus G. roseus G. roseus G. roseus G. roseus G. rubiginosus G. scleroideus G.suboxydans G. suboxydans G. suboxydans G. suboxydans G. suboxydans var. 01 a Number * Viability c Viability d Instihte

IAM 1806 IARI 1813 IAM 1832 IAM 1814 IAM 1816 IAM 1815 IAM 1821 IAM 1822 IAM 1818 IAM 1820 IAM 1841 IAM 1838 IAM 1840 IARI 1839 IAM 1827 IAM 1842 H-15 IAM 1828 G-3 IFO” 3010 IAM 1830 -___--_

108 106 105 108 106 109

2 x 109 7 x 108 3 x 108 108 7 x 109 4 x 108 3 x 108 108 5 x 108 106 108 4 x 108 3 x 105 8 X lo3 3 x 105

of viable cells/ml after storage in lOc7oglycerol after storage at 10” C for 2 years. after storage at 10” C for 2 years. for Fermentation, Osaka.

ence should not be generalized because the viable ‘cell counts were widely scattered reflecting the possible presence of species or strain specificity; also the number of tested strains and taxa were limited.

x x x x x

at -So

/ + + + + i + + + + + +

+ + I_

7 + +

C for 16 months.

Psychrophilic bacteria The strains of psychrophilic bacteria used in this study were those collected in Antarctica and identiiied by Inoue (Table 6). These are all obligate psychrophiles having the optimum tem-

FREEZE-PRESERVATION

A. A. A. A. A. A. A. A.

acrti acrt0s11s ascendem uscendons albwninosuw krcrtzingiantrs mnccns rancens v:~r. I

“Intermediale”

IAM IARI IARI 1AhI IARI IAhl IAM IAM

1802 1804 1808 1811 1807 1817 1825 1826

6 9 5 8 4 2 6

IAJI IA11 1,4hI IAl1

1812 1834 1835 1836

IAM IAM IAM IAM IAM IAM IARI IAM IAM IAM IAM IAM IAhl IA&I IAM

1806 1813 1832 1813 1816 1818 1821 1820 1838 1839 1827 1842 1829 1828 1830

x x x X x x X

109 10’” 10’0 1O’O 10’0 10’0 lo9 10’0

459

OF BACTERIA

107 3 x 10’0 2 x 109 10’0 2 x 109 2 x 109 8 X lo5 4 x 10’

2 x 106 109 3 x 109 6 X lo9 2 x 109 108 9 x 10” 105

4 9 3 8 6 2

JO5 108 107 10G lo7 108 106 2 x 108

3 x 105 108 104 2 X108 2 x 10’ 2 x 108 4 x 10” 5 x 107

5 x 10’0 10” 2 x 10’0 6 X log

10’0 6 X lo9 4 x 109 109

7 6 2 2

x

108 2 x 10’ 9 x 106 2 x 108

3 7 6 3

x x X x

106 106 lo3 107

5 x 109

107 10” 109 109 106 10’” 10’0 109

4 4 5 4 2

x x x x x

2 x 103 4 x 107 10’ 5 x 108 2 x 106 3 x 10’ 3 x 108 9 x 10” 2 x 108 7 x 102 107 4 x 108 5 x 105 5 x 106 :< x 10”

3 9 2 2 2

x x x X x

7 3 3 5 2

x x x x x

103 106 108 lo8 104 10’ 10” 107 107 104 108 105 105 108 105

x x x X X x

St rains

A . aurantirrs G. 1igucfucic:ns G. nudunogenrrs G. rnrlanogenus

x 109 X lo6 x 109 109

Genus Gluconobacter G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.

albidus capsulatus cerinus gluconicus industrius melanogenus melanogcnus melanogenus roseus TOScllS rubiginosus scleroideus suboxydaas suboxydarbs suboxydans WT. (Y

a Number b Number

of viable cells/ml of viable cells/ml

3 x

109

5 x 10’0 8 X lo9 3 x 109 10’” IO” 7 x 109 6 X IO9 2 x 109 10’0 2 x 10’” 2 x 109 6 X lo9 10”

2 2 2 5

x x x x

2 x 109 3 x

2 2 3 2

104

10’0 x 107 x

109

x 108 x 10’0

105 105 106 107 lo” 10’” 7 x 109 2 x 109 109

3 x 103 2 x 109 3 x 10’ 10” 108 6 X lo9

2 x

after storage at -28” C for 4.5 years. afler &wage at 10” C for 4.5 years.

perature of 10” C and the ability to give visible growth at 0” C within 2 weeks. All but three strains were extremely sensitive to frozen storage; three strains gave nil, and the remaining four showed low viability. To attempt to explain cell fragility on the basis of some cell characteristic related to psychrophilicity, such as a different lipid composition of the cell membrane from that of a mesophile, is attractive, but possibly

not feasible. Farrell and Rose (4) reported that a facultative psychrophile of P. aeruginosa was more resistant to freezing temperature than a mesophile of this species. As has been discussed in the previous section, the fragility of P. putrefaciens 65-0-a might be ascribed to some properties specific for this species. Similar to this case of P. putrefaciens, fragility of the strains of the genera of Cytophuga and SpXlum

YAMASATO,

460

OKUNO, AND OHTOMO TABLE

COMPARISON

OF THREE

11

SETS OF THE RESULTS ACETIC

ACID

ON FEOZEN

STORM+E

OF

BXTERIA Eable cells/ml

o lwo

Glycerol -53O c 16 months

1wJ Glycerol -28” c 4.5 years

10% Honey -28” C 4.5 years

Number of strains Proportion (%) Number of strains Proportion (76) Number of strains Proportion (%I) -~-

Tota1

101

102

10”

10’

105 ---

106

10’

108

109

10’0

2

0

0

1

1

5

2

5

15

4

0

35

5.7

0

0

2.9 2.9 14.3

5.7

11.4

0

100

0

00

3

4

12

1

27

0

0

0

11.1 0

0

0

0

0

1

2

0

0

0

0

3.7

7.4

might be specific for the taxa, because mesophilic strains of these genera were stated to be sensitive to freeze-drying and better preserved by liquid-drying (7). Those psychrophilic strains tested here produced a fairly large amount of polysaccharides around the cells which supposedly prevented access for 8cryoprotectants, enforced their preservation under inadequate conditions, and resulted in loss of viability. It is paradoxical that the strains collected in Antarctica and apparently well-adapted to low temperatures are very susceptible to freezing. Lactic acid bacteria. The distribution of the strains at various levels of viability is shown in Table 7. Though all survived frozen storage, the viable cell counts were generally low compared to the above-mentioned aerobic bacteria. Sensitive strains were seen in Lactobacillus delbrueckii, Lactobacillus lactis, Streptococcus lactis, Pediococcus soyae, and Leuconostoc mesenteroides var. sake LY.The viabilities using three preservation methods, namely, freezing, freeze-drying, and under liquid paraffin seal are generally parallel to each other.

0

3

14.3 42.9

2

2

7.4

7.4 44.4

3.7

1

4

1

7

3.7

14.8

11.1 14.8

I1

100

27

3.7 40.7 25.9

100

-~ .~___

This coincides with the results by Kono et al. (6) that the strains sensitive to freezedrying were also sensitive to liquid paraffin seal preservation. Lactic acid bacteria are nutritionally fastidious, and their requirements increase when injured by freezing treatment. Injured cells have been reported to require some components in peptone for recovery (9, 10). Viable cell counts after frozen storage were compared on two kinds of media (Table 8). Medium II is based on Medium I but enriched with various kinds of peptone and additives from commercial sources. The largest difference was observed in the viable cell counts of Lactobacillus helz;eticus IAM 1042 giving a lO,OOO-fold increase on Medium II. The same phenomenon was observed when the strains were revived from the paraffinsealed culture. Some were viable on Medium II but not on Medium I. The reverse case was not observed. Acetic acid bacteria. Acetic acid bacteria very often die after acid production when preserved on agar slants. Moreover, it was reported that their taxonomic character-

FREEZE-PRESERVATION

--

-. Species -..

OF BACTERIA

Struiu

no. ..-.-

..-

3908 6%0-a

-~__

--

Viable -__

~-~ cclls,~‘~nl __

Pseudomonus putrejaciens Pseudomonas putrejaciens

IF0

Acetobacter acetosus Acetobacter acetosns Micrococcus candidus :Ilicrococc~s candidus Staphylococcus epiderwkdis

IAM IA11 IAhI IAM IBM

1805 1804 1433 1422 1296


Nocardia corallina fWocardia corallina Suxina auranl iaca Sarcina albitla Sarcina wzaryinala Aeromonas hydrophila Mycobacterium sp. Bacillus jirmus

3-108 3-7a IA11 1059 IARI 1012 IARI 1130 ATCCh 9071 2-3.Y 20G-1

3 x 104 3 x 102 103 2 x 103 3 x 103 2 x 103 3 x 104 4 x 104

_

--.

ViiLMity

of other

strains

..~.

0 0

a Gaseous hydrocarbon-utilizing bacteria from H. Iizuka, Microbiology, University of Tokyo). h The American Type Culture Collection.

istics sometimes change during preservation by repeated serial transfer. For these reasons long-term preservation is especially desiable. Table 9 shows the viability of these bacteria after three methods of preservation. “Intermediate” strains are those defined by Asai et al. (1) as having characteristics of both Acetobacter and GEuconobacter. Table 10 shows the viable cell counts before freezing, after frozen storage in 10% glycerol, and in 10% honey at -28” C for 4.5 years, and after storage freeze-dried in 10% skim milk + 0.5% Naglutamate and in 5% honey for 4.5 years. The four sets of viability data show similar trends. Honey was previously shown by one of the authors, T. Ohtomo, to be an excellent adjuvant for the freeze-preservation of molds and yeasts ( 11). The three sets of data on frozen storage are compared in Table 11. These bacteria were well preserved for a period as long as 4.5 years, best with honey and next best with glycerol. As in the case of fungi, honey proved also to be an excellent adjuvant for long-term freeze-preservation of

401

>

3 x 108

4 x 107, 3 x 107 >

N. Yeto, and 8. Sakayanagi

2 x 10’

106

(Institute

of

Applied

acetic acid bacteria. But this was not the case with freeze-drying, which might be as a result of the browning reaction during storage. Honey used in the study contained about 80% sugars, of which largely fructose, and amino acids including glutamic acid as minor components. On the assumption that the physical state of water in frozen cells would greatly affect their viability, Gonda et al. (5) estimated the glass transition points of cryoprotectant solutions and the cells soaked in and equilibrated in them by use of differential scanning calorimetry, with the aim of elucidating the effect of the cryoprotectants. Honey solution gave a higher glass transition point than glycerol solution, which was assumed to prevent ice crystal formation within the cells, resulting in better survival of the cells. Since fructose solution gave glass transition points similar to honey solution and the possible role of amino acids has not been clarified, the protection mechanism specifically ascribed to honey components remains yet to be solved.

YAMASATO,

4G2 TABI,E

Group

of bacteria

[Nu~pber strains surviving

OKUNO, AND OHTOMO

13

of /

Number strains tested

of h&r(%) ___-

Aerobic grant-posit,ivos Aerobic gram-neghves Lactic acid l)nct,eria A CClObUClRl “Intermediate” strains Ghconobacter

O!,/x’J .3/O!) 2x,/:::‘, O/IO O/3 5120

84 77 8.5 90 0 25

The viable cell counts after frozen storage at -53” C for 16 months were lower than those at -28” C for 4.5 years. This may be attributed to the difference of the initial cell concentration, freezing procedures, or the extent of temperature deviation which accelerates ice crystal formation during storage. Considering these data and those of aerobic bacteria as well as those in the literature, it should be concluded that it may be possible to preserve a wide variety of bacteria for a long time in the frozen state at moderately low temperatures when appropriate procedures are applied. But there still remains the problem that a small proportion of the strains or species are sensitive and may be difficult to preserve by frozen storage at moderately low temperatures. Possible relationship of species-specificity to tolerance to frozen-storage. Although the number of strains used was limited and many variables affect viability, there seems to be an indication suggesting the influence of species-or strain-specificity on sensitivity to frozen storage (Table 12). The influence of species specificity is indicated in Pseudomonas putrefaciens, Acetobacter acetosus, Micrococcus candidus, and Nocardia corallina. In the righthand column of Table 12 higher viable cell counts of the other strains of the same species are given, suggesting strain specificity. Liquid paraffin seal. The liquid paraffin seal method is simple and in some cases effective for temporary preservation. 75

85% of aerobic bacteria and lactic acid bacteria wcrc kept alive this way. Acetobactcr was well preserved whereas “Intermediate” strains and Gluconobacter were very susceptible (Table 13). The composition of media may partly account for the difference, because Gluconobacter produces more acids on glucose-containing medium. All the strains were alive after both or either of frozen storage at -53” C or paraffin-seal storage. Genus specificity was also observed in viability after paraffin-seal storage. The strains of Bacillus, Co ynebacterium, Arthrobacter, LMicrococcus, Pseudomonas, Aeromonas Achromobacter, and Alcaligenes were generally viable. All three strains of Erwinia herbicola, two strains of Serratia marcestens, two strains of Brevibacterium testaceum, two of three strains of Staphylococcus aureus, four of seven strains of Enterobatter cloacae, and two of six strains of Brevibacterium helvolum were dead after paraffin-seal storage. Of eight strains which showed low viability of O-10* viable cell counts/ml after frozen storage, only one strain was dead and seven strains were alive after paraffin-seal storage for 3.5 years. On the other hand, of 74 strains which showed high viability of 108-1010 viable cell counts/ml after frozen storage, 57 strains were alive and 17 strains were dead. These data are contradictory to results from lactic acid bacteria. Those seven strains which gave low viable cell counts after frozen storage but which were alive after paraffin-seal storage are considered to be sensitive to frozen storage. The application of frozen storage may be unadvisable for these strains. However, they may serve as useful tools for further investigation to improve preservation procedures. SUMMARY

In order to get some basic information for the development of a long-term preservation method by freezing at moderately low temperatures, the viability of 259 strains belonging to 32 genera and 135 spe-

FREEZE-PRESERVATION

ties was measured. Cells were suspended in 10% glycerol and stored at -53” C for 16 months. About 9370, SSS, and 74% of aerobic bacteria gave viable cell counts higher than 105/ml, IOG/ml, and 107/m1, respectively. About 10% of gram-positives and 3% of gram-negatives gave viable cell counts lower than 105/ml. There seemed to be some species-and genus-specificity with respect to viability after frozen storage and liquid paraffin-seal storage. Strains of coryneform bacteria, genera of the family Enterobacteriaceae, and the genus Pscudomonas were generally resistant. Pseudomonas putrefaciens proved to be specifically sensitive. Lactic acid bacteria were subject to sublethal injury, requiring special recovery media. Psychrophilic bacteria were very susceptible to frozen storage. All the tested strains of acetic acid bacteria survived frozen storage well both in 10% glycerol and in 10% honey at -28” C for 4.5 years. Honey proved to be a better adjuvant for frozen storage than glycerol. It was suggested from the results that for many kinds of bacteria, long-term preservation by freezing at moderately low temperatures might be possible when appropriate procedures are applied. ACKNOWLEDGMENT The authors express their thanks to Dr. II. Iizuka, Dr. N. Seto, Mr. S. Sakayanagi, and Mr. K. Inoue for supplying strains and to Mrs. E. Unami and hlrs. Y. Mincmura for useful assistance. REFERENCES 1. Asai, T., Iizuka, II., and Komagata, K. The flagellation and taxonomy of genera Gluconobactcr and i\cetobacter with reference to the existence of intermediate strains. .I. Gen. Appl. Microbial. 10, 95-126 ( 1964). 2. Clement, M. T. Effect of freezing, freeze-drying, and storage in the freeze-dried and frozen state on viability of Escherichia coli cells. Can. 1. Aficrobiol. 7, 99-106 ( 1961).

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3. Eckert, H. L., and Flaherty, D. K. Long-term preservation of Rortletell~ pc2tWssi.s..4ppl. Alicrobiol. 23, 1X6-187 (1972). 4. Farrell, J., and Rose, A. H. Cold shock in a pseudomesophilic and a psychrophilic monad. J. Gen. Microbial. 50, 429439

(19G8). 5. Gonda, K., Ohtomo, T., Yamasato, K., and Koga, S. Thermograms of frozen microbial cells obtained by differential scanning calorimctry. In “Report of Japanese Society for Research of Freezing and Drying.” No. 18, 109-113, 1972. (In Japanese) 6. Kono, K., Nomura, H., and Ozaki, A. Preservation of lactic acid bacteria. In “Report of Japanese Society for Research of Freezing and Drying.” No. 18, 21-25, 1972. (In Japanese ) 7. Lapage, S. P., and Shelton, J. E. Culture collections and the preservation of bacteria. In “Methods in Microbiology” (J. R. Norris and D. W. Ribbons, Eds.), Vol. 3% p. 135. Academic Press, New York, 1970. 8. Mead, D. D., Wessman, G. E., Higuchi, K., and Surgalla, M. J. Stability of cell suspensions of Pasteurella pesti.s at 5C and -23C. Appl. hlicrobiol. 8, 55-60 (1960). 9. Morichi, T. Metabolic injury in frozen Escherichiu coli. In “Freezing and Drying of Microorganisms” (T. Nei, Ed.), pp. 53-68. Univ. of Tokyo Press, Tokyo, 1969. 10. Moss, C. W., and Speck, M. L. Identification of nutritional components in Trypticase responsible for recovery of Eschcrichia coli injured by freezing. .I. Bocteriol. 91, 10981104 (19G6). 11. Ohtomo, T. Protective substances for freezing and drying of fungi. In “Protective Substances for Freezing and Drying” (T. Nei, Ed.), pp. 121-133, Univ. of Tokyo Press, Tokyo, 1971. (In Japanese) 12. Servin-Mass& M., and Cruz-Camarillo, R. Variants of Serratia marcescens induced by freeze-drying. Appl. Microbial. 18, G89G91 (19G9). 13. Webb, S. J. Some effects of dehydration on the genetics of microorganisms. In “Freezing and Drying of Microorganisms” (T. Nei, Ed.), pp. 153-167. Univ. of Tokyo Press, Tokyo, 1969. 14. Yurchenco, J. A., Piepoli, C. R., and Yurchenco, M. C. Low temperature storage for maintaining stable infectious bacterial pools.

AppZ. Aficrobiol. 2, 53-55 (1954).