Effect of freezing and thawing on survival of three bacterial isolates from an arctic soil L O U I S EM. NELSON^
AND
D. PAKKINSON
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/14/15 For personal use only.
I~c~pot.!ttrc~trr c!/'Biolo,y~. Utrit~er:si!y c~/'Crrlgrrt:\.,Crrlgrrr:\~,dl!rr., Crrtrrtrln 7'2N I N 4 Accepted August 9. 1978 N . EtTect of freezing and thawing on sul-vival of three NELSON,L. M.. and D. P A R K I N S O 1978. bactel-ial isolates f~.oman arctic soil. Can. J . Microbiol. 24: 1468-1474. Three isolates, :I P.cc~cctlottiotrcr.~ sp..Boc.illti.v sp.. itnd Artlrro1)trc~trr~sp.. which h;ld been isolated from a meadow soil at Devon Island, C;~n;tcln.were subjected to freezing and thawing at low rates undel. various conditions. When cells were frozen in sand or soil. survival was dependent on moistu~.elevel. storage time. and thaw rate. P.s~~rtlot~iotrtt.s M216 was most susceptible to freeze-thaw dam;~ge~ ~ n d these er conditions. Ar!lrrohrrc~/~rM j I was the most resistant o f the three isolates when frozen in sand or soil and when frozen ;tt ;I high rate after growth at vtrring rates ;it 5 and 15°C in C;II-bon-o r nitrogen-limited media. P.s~rrtln~trotrrr.s M216 was more sensitive to freeze-thaw damage when NnCI was present in the freezing menstruum, even at low freezing rates. Survival of cells fi-ozen in growth rnedilirn. water, saline. :uid soil extract was not affected by the freezing rate when i t was less than 1°C niin I . Soil extract did not protect cells fi.om freeze-thaw damage any more than water and for Artllrohoc./o. MSI survival was decreased when i t was frozen in soil extract. NELSON.L. M . . et D. P A R K I N S O N 1978. . Effect of freezing and thawing on s u ~ ~ v i vof ~ t three l bacter-ial isolates fr-om an arctic soil. Can. J . Microbiol. 24: 1468-1474. Nous avons sournis it d e fi~iblest:t~~x tle congelation et de decongel;ttion sous diverses condiBrrc~illris.et Ar!lrrohrrt~!~~~: ils ont ete tions, trois isol;tts appartenant aux genres Ps~~rtlot~iotrtr,s. isoles d'un sol cle prairie de I'ile Devon all Canada. Lorsque les cellules sont congelees dans le sable ou clans le sol. la survie depend du degre d'hurnidite. du temps d e conhervation et de la M2Ihest pltls sensible ; I L I ~dommoges causes decongelation. Dans ces conditions. P.sc~~rtlottrotrrt,s par la congelation et la decungPlation. Ar!lrt.obtrcto M j l cst le plus resistant des trois isolitts lorsqtl'il est congele dans le sable ou le sol. ninsi que lorsque la congelation est tres elevee. apres qu'il se soit developpe it divers tails de croiss:tnce it 5 et 1S0Cdans on milieu limite en c t ~ ~ . b o et ne en azote. Ps~trtlor~roirct.sMa16 est plus sensible aux domrnnges de la congelation et d e la decongelation lorsque NaCI est present d:tns le solvent de congelation, et ce meme p o u r d e s faux decongelation bas. La survie des cellules congelees clans le milietlde cl-oissiince, I'eau, les s e l h o t ~ I'ext~.;ritde sol n'est pas affectee par le taux de congelation lorsque celui-ci est infcrieur :I 1°C min-I. L'extrait cle sol ne protkge pus plus les cellules des dommages d e la congelation et de la deco11gil:ttion que I'e:ul. et lit sul-vie d',4t.t/1robtrc,tc,r M j I decroit quand i l est conge16 dans de I'ext~'aitde sol. [Traduit par le jou~.n;~ll
Introduction Microorganisms in soils from temperate and polar regions are subject to periods of freezing and thnwing several times a year. Freezing and thawing of soil acts as a physical weathering agent and can also lead to a release of va~.ious cations (Summerfield and Rieley 1973), amino acids (Ivarson and Sowden 1966), and sugars (Ivarson and Gupta 1967). Freezing and thawing of soils under label-atory conditions led to a decrease in the number of soil bacteria (Biederbeck and Campbell 1971) which, it was postulated, might provide a ready source of nutrients for the surviving microorganisms (Campbell and Biederbeck 1972; Biederbeck and Campbell 1973). Because of the short growing season and low nutrient levels of tundra soils, spring thaw may represent a crucial time for 'Present address: Department of Agricult~~ral Science. University of Oxford, Parks Ro;td. Oxford. England OX1 3PF.
growth of soil microorganisms and a peak in bacterial numbers was observed during spring thaw at Signy Island (Baker 1970) and at Barrow, Alaska (Bovd . , 1958: Bunnell et 01. 1975). The subsesuent rapid decline observed after thaw might be attributed to the deleterious effects of thawing at fluctuating tempelxtul-es as seen in the label-atol-y (Biederbeck and Campbell 1971). Resistance to freeze-thaw stress is likely, therefore, to be an important factor in the successful competition of tundra soil bacteria. T h e mechanisms leading to injury and death of a microbial cell as a result offreezing and thawing are not yet completely understood, but at least two frictors seem t o be involved which are oppositely dependent on cooling rate (Mazur 1970). Many factors can influence the response of bacteria to freezing and thawing; 13 of these were reviewed by MacLeod and Calcott ( 1976). They observed a general pattern of response to freezing and thawing in
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/14/15 For personal use only.
NELSON A N D P A R K I N S O N
which cell survival increased as cooling rate increased and then decreased to a minimum before increasing again at ~lltrarapidcooling rates. The warming rate affected sul.vival only at high cooling rates. A numbel- of f>ictorscould affect this wattern while the type of damage pl-oduced was dependent on the composition of the suspending fluid (Calcott and MacLeod 1974~1,1974b, 1975~1,19756). Studies of this sort have been important in elucidating the mechanisms leading to cell damage and death as a result of freezing and thawing but the freezing rates used in these studies of Mazur and Calcott and MacLeod were generally greater than 1°C per minute, an ~~nnaturally high rate for organisms in soil. Little information is available on sul-vival of organisms indigenous to soil or of those from cold environments. As part of a study of the growth and survival characteristics of three bacterial isolates from an arctic soil, we investigated the resistance of these isolates to freezing and thawing at low freezing rates and the influence on survival of a number of factors including composition of the freezing menstruum, growth conditions prior to fleezing, and rates of cooling and warming. The results of these studies are presented here. Methods Orgtr~ri.s~~r.c Three isol;~tesfrom the hummockv sedge - moss meadow site at Truelove Lowland, Devon ~ s l a n i N.W.T. , (75"33' N , 84"401 W) were designated as P.scrrt1o11lo11ci.sM216, Bac~illri,sM 153, and Artl~robcrcler M5 I. Pserrtlo~~rorrtr.s M 2 16 and A~.tlrrobcictr~M5 1 were psychrotrophic and representative o f genera commonly isolated from the nieadow site, while the Bacil1rr.s isolate was more mesophilic and occurred infrequently. Taxonomy, nutrition, and growth ch:u-acteristics o f these isolates were described by Nelson and Parkinson (1978) and a detailed site description wasgiven by Bliss (1975). The isolates were maintained at 5°C in semisolid peptone yeast extract agar. . .
. , . .
.
,
F r r o - i ~ ~~gI Strl~rl I r i ~ l dSoil Cultures ofeach isolate were grown in medium K (Nelson and Parkinson 1978) supplemented with 0.1% yeast extract at 15°C. Cells were harvested in Inte exponential phase, washed, and then resuspended in 0.1 M phosphate buffer. These suspensions were added to screw cap test tubes containing 5Og sterile, acid-washed sand or 0.5 g sterile. air-dried, sieved soil fi-om the meadow site, Devon Island, to provide adensity o f lo7 cells per gl-am ofsoil or sand. The sand had been sterilized b y autoclaving and the soil by ethylene oxide for 180min. The tubes were subjected to two treatments: in one group the soil or sand was saturated with sterile water and in the second, the subst~.atewas moistened only by the inoculum, 0.15 ml in the sand-containing tubes and 0.5 ml in the soil-containing tubes. Soil-mstric potentials o f each treatment were determined b y the pressuremembrane method (Richards 1965). Slow freezing was accomplished by holding the tubes for 24 h successively at each o f the following temperatures: 3, 0, -5, - 15, and -22°C. Thawing was carried out immediately after freezing or after 3 months' storage at -22'C b y placing the tubes in 21 3°C cold room for 4 h (quick thaw). Stored tubes were also thawed b y reversing the freezing treatment (slow thaw). After
1469
thawingquickly or slowly to 3"C, the contentsofeach tube were diluted with i strength peptone yeast extract medium (Nelson and Parkinson 1978) at 20°C and spread onto agar plates o f the medium, The plates at 200C for I week prior to counting, There were two to three replicates per treatment and the mean values are reported. Results were exoressed as the percentage o f colonieh occurring before freezing after equilibration for24 h ;it 3°C. Concurrently, unsterilized, air-dried, sieved soil at two mnt1.i~ potentials wa\ subjected to the freeze-thaw tre~ltmentsdescribed above and the viability o f the indigenous bacterial population was assessed. Ejyect of Cro~t,tlrC o ~ r t l i t i o ~prior r s to Freeze-Tlrtr n) 0 1 1 Srrr~,i~ltrl Duplicate 2-ml aliquots were taken from each carbon- and nitrogen-limitecl continuous-culture esperiment at 15 and 5°C as described by Nelson (submitted for publication), frozen at -70°C fo1.24-48 h. thawed at 20°C and viability assessed by the slide-culture method at 20°C as described by Nelson (submitted for publication). The freezing and thawing rates were equiv:rlent to 150°C h--' and 420°C h- I , respectively. Srrrr.i~~crl (11 Vo,:\.i~rjiF r e e z i ~ ~crlrtl g Tlrcr~r,i~rg Rrrtes i ~ rG r o \ ~ ~ l i Me(1ir11t1 Duplicate 2-ml aliquots, from nitrogen-limited continuous cultures ofeach isolirte at a dilution rateof 0.04 h ' at 5°C. were frozen at five I-ates from 3°C h-' to 60°C h 1 in ;I Haake thermostat K T 5 2 coupled with the programmer PG I I. Freeze-thaw rates were monitored with n YSI thel-mistor probe, telethermometer(model42). and a recorder. These samples were frozen in the Devon to -20°C, the approximate mid-wintertenipe~xture Island meadow (Courtin and Goodwin, unpublished data). Thawing was fast 01-slow, the former b y dilution with equal volumes o f distilled water at 20°C (180°C h - ' ) and the latter by the slide-culture method at 20°C as previously described. S i ~ i ~ ~ t~i t i ~Voryi~rg ~ t r l F r e r z i ~ ~trtrtl g Tlrcnvi~~g Rtrtes i ~ rWtrter. S a l i ~ r ecr~id , Soil E . ~ t r t ~ c t T o detel-mine the effects o f water, NaCI, and soil extract on survival o f freezing and thawing, aliquots from carbon-limited continuous cultures o f each isolate @own at 0.02 h ' dilution rate at 5°C) were washed twice in distilled water and resuspended in duplicate in distilled water, 0.85% saline, and soil extract prior to freezing at five rates in the Haake thermostat and thawing at 180°C h-I as descr-ibed above. Soil extract was prepared by autoclaving lOOg Devon Island meadow soil in 1000 ml distilled water and filtering through0.2-pm Millipore membrane filters. Viability was detei-mined by the slide-culture method at 20°C as previously described. T o determine whether the survival ofcells in the three media was similar throughout the cooling curve, cells were frozen at 30°C h-I, samples taken as six intervals between 5 and -38°C (the lower limit for linear cooling rates in the Haake thermostat), thawed at 180°C h-I, and viability assessed by the slide-culture method at 20°C as previously described.
Results Effect of Freezing ( ~ t Tlzn~ving ~d oti SLI~-viva1 in Srrnd ~ l n dSoil Survival after freezing and thawing of the three isolates inoculated into sand and soil was enhanced as the water availability decreased (decreasing matric potential) (Table 1). Storage for 3 months at -22°C prior to thawing led to a decrease in the survival of organisms frozen at high matric potential. A slow thaw rate was more deleterious to
C A N . J. MICROBIOL. VOL. 1 4 . 1978
TABLE1. Viability (4,of initial population) of bacteria after freezing to -22'C and thawing in soil and sand at various moisture levels. The effects of storage at -22'C for 3 months and a fast- or slow-thaw rate on survival were determined
4, viability ( k standard error)
v"
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/14/15 For personal use only.
Treatment
Pserrdot17ona.s M216
Bacillris M153
AND
D. PAKKINSON
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/14/15 For personal use only.
I~c~pot.!ttrc~trr c!/'Biolo,y~. Utrit~er:si!y c~/'Crrlgrrt:\.,Crrlgrrr:\~,dl!rr., Crrtrrtrln 7'2N I N 4 Accepted August 9. 1978 N . EtTect of freezing and thawing on sul-vival of three NELSON,L. M.. and D. P A R K I N S O 1978. bactel-ial isolates f~.oman arctic soil. Can. J . Microbiol. 24: 1468-1474. Three isolates, :I P.cc~cctlottiotrcr.~ sp..Boc.illti.v sp.. itnd Artlrro1)trc~trr~sp.. which h;ld been isolated from a meadow soil at Devon Island, C;~n;tcln.were subjected to freezing and thawing at low rates undel. various conditions. When cells were frozen in sand or soil. survival was dependent on moistu~.elevel. storage time. and thaw rate. P.s~~rtlot~iotrtt.s M216 was most susceptible to freeze-thaw dam;~ge~ ~ n d these er conditions. Ar!lrrohrrc~/~rM j I was the most resistant o f the three isolates when frozen in sand or soil and when frozen ;tt ;I high rate after growth at vtrring rates ;it 5 and 15°C in C;II-bon-o r nitrogen-limited media. P.s~rrtln~trotrrr.s M216 was more sensitive to freeze-thaw damage when NnCI was present in the freezing menstruum, even at low freezing rates. Survival of cells fi-ozen in growth rnedilirn. water, saline. :uid soil extract was not affected by the freezing rate when i t was less than 1°C niin I . Soil extract did not protect cells fi.om freeze-thaw damage any more than water and for Artllrohoc./o. MSI survival was decreased when i t was frozen in soil extract. NELSON.L. M . . et D. P A R K I N S O N 1978. . Effect of freezing and thawing on s u ~ ~ v i vof ~ t three l bacter-ial isolates fr-om an arctic soil. Can. J . Microbiol. 24: 1468-1474. Nous avons sournis it d e fi~iblest:t~~x tle congelation et de decongel;ttion sous diverses condiBrrc~illris.et Ar!lrrohrrt~!~~~: ils ont ete tions, trois isol;tts appartenant aux genres Ps~~rtlot~iotrtr,s. isoles d'un sol cle prairie de I'ile Devon all Canada. Lorsque les cellules sont congelees dans le sable ou clans le sol. la survie depend du degre d'hurnidite. du temps d e conhervation et de la M2Ihest pltls sensible ; I L I ~dommoges causes decongelation. Dans ces conditions. P.sc~~rtlottrotrrt,s par la congelation et la decungPlation. Ar!lrt.obtrcto M j l cst le plus resistant des trois isolitts lorsqtl'il est congele dans le sable ou le sol. ninsi que lorsque la congelation est tres elevee. apres qu'il se soit developpe it divers tails de croiss:tnce it 5 et 1S0Cdans on milieu limite en c t ~ ~ . b o et ne en azote. Ps~trtlor~roirct.sMa16 est plus sensible aux domrnnges de la congelation et d e la decongelation lorsque NaCI est present d:tns le solvent de congelation, et ce meme p o u r d e s faux decongelation bas. La survie des cellules congelees clans le milietlde cl-oissiince, I'eau, les s e l h o t ~ I'ext~.;ritde sol n'est pas affectee par le taux de congelation lorsque celui-ci est infcrieur :I 1°C min-I. L'extrait cle sol ne protkge pus plus les cellules des dommages d e la congelation et de la deco11gil:ttion que I'e:ul. et lit sul-vie d',4t.t/1robtrc,tc,r M j I decroit quand i l est conge16 dans de I'ext~'aitde sol. [Traduit par le jou~.n;~ll
Introduction Microorganisms in soils from temperate and polar regions are subject to periods of freezing and thnwing several times a year. Freezing and thawing of soil acts as a physical weathering agent and can also lead to a release of va~.ious cations (Summerfield and Rieley 1973), amino acids (Ivarson and Sowden 1966), and sugars (Ivarson and Gupta 1967). Freezing and thawing of soils under label-atory conditions led to a decrease in the number of soil bacteria (Biederbeck and Campbell 1971) which, it was postulated, might provide a ready source of nutrients for the surviving microorganisms (Campbell and Biederbeck 1972; Biederbeck and Campbell 1973). Because of the short growing season and low nutrient levels of tundra soils, spring thaw may represent a crucial time for 'Present address: Department of Agricult~~ral Science. University of Oxford, Parks Ro;td. Oxford. England OX1 3PF.
growth of soil microorganisms and a peak in bacterial numbers was observed during spring thaw at Signy Island (Baker 1970) and at Barrow, Alaska (Bovd . , 1958: Bunnell et 01. 1975). The subsesuent rapid decline observed after thaw might be attributed to the deleterious effects of thawing at fluctuating tempelxtul-es as seen in the label-atol-y (Biederbeck and Campbell 1971). Resistance to freeze-thaw stress is likely, therefore, to be an important factor in the successful competition of tundra soil bacteria. T h e mechanisms leading to injury and death of a microbial cell as a result offreezing and thawing are not yet completely understood, but at least two frictors seem t o be involved which are oppositely dependent on cooling rate (Mazur 1970). Many factors can influence the response of bacteria to freezing and thawing; 13 of these were reviewed by MacLeod and Calcott ( 1976). They observed a general pattern of response to freezing and thawing in
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/14/15 For personal use only.
NELSON A N D P A R K I N S O N
which cell survival increased as cooling rate increased and then decreased to a minimum before increasing again at ~lltrarapidcooling rates. The warming rate affected sul.vival only at high cooling rates. A numbel- of f>ictorscould affect this wattern while the type of damage pl-oduced was dependent on the composition of the suspending fluid (Calcott and MacLeod 1974~1,1974b, 1975~1,19756). Studies of this sort have been important in elucidating the mechanisms leading to cell damage and death as a result of freezing and thawing but the freezing rates used in these studies of Mazur and Calcott and MacLeod were generally greater than 1°C per minute, an ~~nnaturally high rate for organisms in soil. Little information is available on sul-vival of organisms indigenous to soil or of those from cold environments. As part of a study of the growth and survival characteristics of three bacterial isolates from an arctic soil, we investigated the resistance of these isolates to freezing and thawing at low freezing rates and the influence on survival of a number of factors including composition of the freezing menstruum, growth conditions prior to fleezing, and rates of cooling and warming. The results of these studies are presented here. Methods Orgtr~ri.s~~r.c Three isol;~tesfrom the hummockv sedge - moss meadow site at Truelove Lowland, Devon ~ s l a n i N.W.T. , (75"33' N , 84"401 W) were designated as P.scrrt1o11lo11ci.sM216, Bac~illri,sM 153, and Artl~robcrcler M5 I. Pserrtlo~~rorrtr.s M 2 16 and A~.tlrrobcictr~M5 1 were psychrotrophic and representative o f genera commonly isolated from the nieadow site, while the Bacil1rr.s isolate was more mesophilic and occurred infrequently. Taxonomy, nutrition, and growth ch:u-acteristics o f these isolates were described by Nelson and Parkinson (1978) and a detailed site description wasgiven by Bliss (1975). The isolates were maintained at 5°C in semisolid peptone yeast extract agar. . .
. , . .
.
,
F r r o - i ~ ~~gI Strl~rl I r i ~ l dSoil Cultures ofeach isolate were grown in medium K (Nelson and Parkinson 1978) supplemented with 0.1% yeast extract at 15°C. Cells were harvested in Inte exponential phase, washed, and then resuspended in 0.1 M phosphate buffer. These suspensions were added to screw cap test tubes containing 5Og sterile, acid-washed sand or 0.5 g sterile. air-dried, sieved soil fi-om the meadow site, Devon Island, to provide adensity o f lo7 cells per gl-am ofsoil or sand. The sand had been sterilized b y autoclaving and the soil by ethylene oxide for 180min. The tubes were subjected to two treatments: in one group the soil or sand was saturated with sterile water and in the second, the subst~.atewas moistened only by the inoculum, 0.15 ml in the sand-containing tubes and 0.5 ml in the soil-containing tubes. Soil-mstric potentials o f each treatment were determined b y the pressuremembrane method (Richards 1965). Slow freezing was accomplished by holding the tubes for 24 h successively at each o f the following temperatures: 3, 0, -5, - 15, and -22°C. Thawing was carried out immediately after freezing or after 3 months' storage at -22'C b y placing the tubes in 21 3°C cold room for 4 h (quick thaw). Stored tubes were also thawed b y reversing the freezing treatment (slow thaw). After
1469
thawingquickly or slowly to 3"C, the contentsofeach tube were diluted with i strength peptone yeast extract medium (Nelson and Parkinson 1978) at 20°C and spread onto agar plates o f the medium, The plates at 200C for I week prior to counting, There were two to three replicates per treatment and the mean values are reported. Results were exoressed as the percentage o f colonieh occurring before freezing after equilibration for24 h ;it 3°C. Concurrently, unsterilized, air-dried, sieved soil at two mnt1.i~ potentials wa\ subjected to the freeze-thaw tre~ltmentsdescribed above and the viability o f the indigenous bacterial population was assessed. Ejyect of Cro~t,tlrC o ~ r t l i t i o ~prior r s to Freeze-Tlrtr n) 0 1 1 Srrr~,i~ltrl Duplicate 2-ml aliquots were taken from each carbon- and nitrogen-limitecl continuous-culture esperiment at 15 and 5°C as described by Nelson (submitted for publication), frozen at -70°C fo1.24-48 h. thawed at 20°C and viability assessed by the slide-culture method at 20°C as described by Nelson (submitted for publication). The freezing and thawing rates were equiv:rlent to 150°C h--' and 420°C h- I , respectively. Srrrr.i~~crl (11 Vo,:\.i~rjiF r e e z i ~ ~crlrtl g Tlrcr~r,i~rg Rrrtes i ~ rG r o \ ~ ~ l i Me(1ir11t1 Duplicate 2-ml aliquots, from nitrogen-limited continuous cultures ofeach isolirte at a dilution rateof 0.04 h ' at 5°C. were frozen at five I-ates from 3°C h-' to 60°C h 1 in ;I Haake thermostat K T 5 2 coupled with the programmer PG I I. Freeze-thaw rates were monitored with n YSI thel-mistor probe, telethermometer(model42). and a recorder. These samples were frozen in the Devon to -20°C, the approximate mid-wintertenipe~xture Island meadow (Courtin and Goodwin, unpublished data). Thawing was fast 01-slow, the former b y dilution with equal volumes o f distilled water at 20°C (180°C h - ' ) and the latter by the slide-culture method at 20°C as previously described. S i ~ i ~ ~ t~i t i ~Voryi~rg ~ t r l F r e r z i ~ ~trtrtl g Tlrcnvi~~g Rtrtes i ~ rWtrter. S a l i ~ r ecr~id , Soil E . ~ t r t ~ c t T o detel-mine the effects o f water, NaCI, and soil extract on survival o f freezing and thawing, aliquots from carbon-limited continuous cultures o f each isolate @own at 0.02 h ' dilution rate at 5°C) were washed twice in distilled water and resuspended in duplicate in distilled water, 0.85% saline, and soil extract prior to freezing at five rates in the Haake thermostat and thawing at 180°C h-I as descr-ibed above. Soil extract was prepared by autoclaving lOOg Devon Island meadow soil in 1000 ml distilled water and filtering through0.2-pm Millipore membrane filters. Viability was detei-mined by the slide-culture method at 20°C as previously described. T o determine whether the survival ofcells in the three media was similar throughout the cooling curve, cells were frozen at 30°C h-I, samples taken as six intervals between 5 and -38°C (the lower limit for linear cooling rates in the Haake thermostat), thawed at 180°C h-I, and viability assessed by the slide-culture method at 20°C as previously described.
Results Effect of Freezing ( ~ t Tlzn~ving ~d oti SLI~-viva1 in Srrnd ~ l n dSoil Survival after freezing and thawing of the three isolates inoculated into sand and soil was enhanced as the water availability decreased (decreasing matric potential) (Table 1). Storage for 3 months at -22°C prior to thawing led to a decrease in the survival of organisms frozen at high matric potential. A slow thaw rate was more deleterious to
C A N . J. MICROBIOL. VOL. 1 4 . 1978
TABLE1. Viability (4,of initial population) of bacteria after freezing to -22'C and thawing in soil and sand at various moisture levels. The effects of storage at -22'C for 3 months and a fast- or slow-thaw rate on survival were determined
4, viability ( k standard error)
v"
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by McMaster University on 01/14/15 For personal use only.
Treatment
Pserrdot17ona.s M216
Bacillris M153
