Methylcellulose - Applied and Environmental Microbiology

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Aug 3, 1981 - 42, No. 5. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1981, p. 872-877. 0099-2240/81/1 10872-06$02.00/0. Bacterial Culture ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1981, p. 872-877 0099-2240/81/1 10872-06$02.00/0

Vol. 42, No. 5

Bacterial Culture Preservation in Frozen and Dry-Film Methylcellulose TREVOR V. SUSLOW* AND MILTON N. SCHROTH Department of Plant Pathology, University of California, Berkeley, California 94720 Received 9 December 1980/Accepted 3 August 1981

Forty-seven of 61 bacterial cultures, including strains of Pseudomonas, Xanthomonas, Erwinia, Agrobacterium, Corynebacterium, Serratia, Klebsiella, and Escherichia, remained viable after storage in frozen methylcellulose or in dried methylcellulose for up to 38 months. Pathogenicity remained intact for those strains tested. Bacteria were grown on a solid medium and then removed and placed in 1.0% methylcellulose (cellulose methyl ether) to make a final suspension of 10' colony-forming units (CFU) per ml. For storage in dried form, the bacteriamethylcellulose suspension was placed in a petri dish and dried in a forced-air incubator. After 24 h of storage at 25°C, viable populations of 105 CFU/mg (equivalent to 106 CFU/ml) were recovered. Populations of 102 to 104 CFU/mg were recovered after storage of up to 38 months. Similar results were obtained in frozen methylcellulose. Survival was greatly enhanced when the growth medium for the bacteria was potato dextrose peptone rather than nutrient agar, yeast dextrose calcium carbonate peptone, or King's medium B. Addition of 0.1 M MgSO4 to the methylcellulose suspension and to the resuspending liquid also increased survival and recovery from storage for some strains. Methylcellulose storage should be a simple, inexpensive, and reliable method of maintaining cultures for short or long periods of time.

While developing techniques to apply specific plant growth-promoting bacteria to seeds in a powder form (T. Suslow, Ph.D, thesis, University of California, Berkeley, 1980), we observed that cellulose methyl ether (methylcellulose; MC) is an effective menstruum for storing bacteria for both short and long periods of time. Initial experiments suggested that it could be used as an alternative to lyophilization and liquid nitrogen storage and should be especially useful for small laboratories interested in a fast, simple, inexpensive, but reliable method to preserve bacteria. This paper reports the development of methods using frozen and dry-film MC to store bacteria. A preliminary report has been published (20). some strains (3, 6, 13, 18). Low and ultra-low MATERIALS AND METHODS (liquid N2) temperature storage also have been used effectively (3, 5, 12, 15, 17). Among the Preparation of MC and comparison to other numerous materials and methods used for pre- water-soluble polymers. The technique of preparserving bacterial cultures are sterile water (22), ing MC (cellulose methyl ether [4,000 cps]; Mallinckphosphate buffer (21), sucrose (4), dextran (4), rodt Chemical Works, St. Louis, Mo.) in suspension mineral oil (4), glycerol (3, 8, 15-17), dimethyl was critical for its effective use as a bacterial preservwas simple and reliable if the sulfoxide (6), polyvinyl pyrrolidone (6), poly- ative. The technique were followed. Prior tests had procedures following acrylamide gels (5), and, most recently, anhy- shown that a 1.0% suspension was the most effective drous silica gel (6, 9, 18, 21). The effectiveness of for bacterial survival. Suspensions of 1.0% MC (wt/ each material depends greatly on the bacterial vol) were prepared by adding, with stirring, MC powstrain and the duration and conditions of storage der to distilled water. The suspension was slowly (11, 12, 19). brought to a boil and boiled for 5 to 10 min until small The preservation of bacterial stock cultures to maintain viability and biochemical or virulence characteristics is an integral requirement for the continuity of microbiological research. Shortterm and long-term storage techniques are employed routinely because bacterial strains often lose desired properties or characteristics after repeated transfer on culture media. Numerous microbial preservation techniques are available, but these generally require extensive preparation of the storage menstrum and expensive equipment for freeze-drying or low-temperature maintenance of cultures. Lyophilization (2,8,22) is generally accepted as the most reliable storage method, although mutations, loss of virulence, and loss of viability during storage occur with

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BACTERIAL PRESERVATION IN METHYLCELLULOSE

amorphous aggregates of MC formed. Unless boiling temperature is attained, since MC does not stay in suspension, this preparation will be unsatisfactory for use as a bacterial preservative. The MC suspension was then autoclaved for 15 min at 121 lb/in2, which caused it to become a colloidal gel. Upon cooling, the MC gel dispersed and remained in suspension. The final pH was 7.2. Three other water-soluble polymers, hydroxethylcellulose, polyvinyl alcohol, and carboxymethyl cellulose, were tested for use as culture preservatives. Comparisons of all four polymers were made by creating dried films (described below) of Erwinia carotovora subsp. carotovora and Pseudomonas marginalis pathovar (pv.) marginalis at 28°C and determining viable populations after 1 week and 1 month. MC was the most effective storage menstruum, and results for other materials are not presented. Preparation of bacterial strains for frozen storage. Bacterial strains were grown on King medium B (KB) for 48 to 72 h at 28°C. Cells were subsequently washed off medium plates into sufficient 1.0% MC solution to form a final suspension of 108 colony-forming units (CFU) per ml. Three milliliters of the suspension was placed into a sterile 5-ml screwcapped vial and frozen in a standard refrigerator freezer at -14°C. Other storage temperatures tested for some strains were 25 and 4°C. Vials were thawed by removing them from the freezer and incubating at room temperature. Three 1-nil samples were taken from each vial at various intervals ranging from 24 h to 38 months and plated on medium to determine viability. Unless otherwise stated, all reisolations were made by dilution series plating with sterile distilled water on KB. The effect of periodic freezing, thawing, and refreezing on the survival of the bacteria was tested. Strains of Serratia marcescens, E. carotovora subsp. carotovora, Xanthomonas campestris pv. begoniae, Pseudomonas syringae pv. syringae, and Pseudomonas fluorescens were prepared for frozen storage, as described, and 10-ml samples of the bacterial MC solution were placed in 20-ml screw-capped glass vials. Individual vials were removed from freezer storage, thawed, sampled, and refrozen at 10-day intervals for 40 days. Preparation of bacterial strains for storage in dry-film MC. Bacterial suspensions of 108 CFU/ml in 1.0% MC were prepared as described for frozen storage. Fifteen milliliters of a bacterial MC suspension was placed in a plastic petri dish (85-mm diameter) and dried in a forced-air incubator at 10, 24, 28, or 32°C. Drying time was approximately 15 h at lower temperatures and 9 h at 28°C. A thin film weighing approximately 60 mg was formed with a residual moisture content of 12.7%. Bacterial viability in stored film was tested at various intervals up to 38 months by aseptically removing 1-cm2 sections of film and placing in 10 ml of sterile distilled water. Samples in sterile distilled water were intermittently agitated on a rotary shaker for 10 min until they were resuspended. Population determinations were made on KB. Bacterial strains stored in MC. Sixty-one bacterial isolates, including representative strains of Agrobacterium, Corynebacterium, Erwinia, Esche-

873

richia, Klebsiella, Pseudomonas, Serratia, and Xanthomonas, were tested for viability, pathogenicity, or other biological properties after storage in frozen or dry-film MC. Bacterial strains evaluated for survival in these studies were obtained directly from plant material or from our bacterial culture collection. Strains from the culture collection had previously been stored as lyophilized cultures, in half-strength nutrient broth, or in sterile tap water. A list of strains used in these tests is presented in Tables 1 and 2. Pathogenicity of plant pathogenic bacteria was tested by standard wound inoculations of appropriate host plants or by hypersensitive reaction on tobacco (Nicotianaglutinosa var. Glurk) (10) or by both methods. The biological activity of Agrobacterium radiobacter 84 and plant growth-promoting rhizobacteria were tested by in vitro antibiosis tests (14, 20). A. radiobacter 84 was also tested for maintenance of in planta antibiosis against Agrobacterium tumefaciens C58 on tomato (14). Plant growth-promoting strains Pseudomonas putida SH5 and B4 and P. fluorescens RV3 were inoculated onto sugar beet and evaluated for plant growth enhancement and antibiosis towards fungal and bacterial plant pathogens according to described procedures (Suslow, thesis). Effect of growth media on bacterial survival. Four standard bacteriological media were compared for their effect on survival of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, A. radiobacter 84, and A. tumefaciens in MC. Media compared were KB, nutrient agar, potato dextrose peptone, and yeast dextrose calcium carbonate peptone. Bacterial strains were incubated at 28°C for 72 h and then washed from each medium into 0.1 M MgSO4 and placed in a 1.0% MC solution. Bacterial suspensions were adjusted to approximately 109 CFU/m1. Initial populations for each suspension were determined on KB by dilution series plating. Samples of each bacterial strain from all source media were prepared for frozen or dry-film storage as described. Surviving populations of each strain were. determined at various intervals up to 8 months by dilution plating on KB. Effect of MgSO4 on increasing bacterial recovery from MC. On the basis of several reports (12, 15, 17) which claimed that Mg2" enhanced bacterial recovery from freezing and desiccation stress, MgSO4 was tested for its effect on increasing the survival and recovery of Pseudomonas phaseolicola HB36 and A. radiobacter 84 from frozen and dry-film MC. Five replications were prepared in sterile distilled water, and five were prepared in 0.1 M MgSO4. An additional five vials with autoclaved 0.1 M MgSO4 adjusted to 108 CFU/ml for each strain were frozen form comparison to MC. Recovery from storage after 6 weeks was determined by dilution plating on KB.

RESULTS During the course of the study, several factors such as culture medium and addition of Mg2+ to the storage. medium were discovered that reduced the death rate of some bacteria. The original MC technique was therefore modified during the investigation to incorporate these new improvements. Although data from the im-

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APPL. ENVIRON. MICROBIOL.

TABLE 1. Survival periods of bacterial strains stored in frozen and dry-film MC Frozen

Bacterial strain

Time in

C58 Corynebacterium fascians 715 C. flaccumfaciens pv. flaccumfaciens 729 C. michiganese pv. michiganese 746 Erwinia amylovora E. carotovora subsp. atroseptica subsp. betavasculorum

subsp. carotovora E. chrysanthemi P-i 14a

14b

Escherichia coli Klebsiella spp. Pseudomonas aeruginosa P. cepacia 459 P. fluorescens RV3 481

P. marginalis SL-5 544

Fi

a

storagea

38

3.1 x 105

36

9.7 x 102

38 38 22 22 38 38 38 38

1.9 x 9.8 x 3.7 x 2.0 x 8.0 x 5.1 x 5.0 x 7.8 x

36 26 26 26 26 26 32 *32

3.7 x 102 1.8 x 102 32 2.9 x 102 89 79 1.7 x 103 1.7 X 102

38 38 38

2.9 x 103 7.0 x 104 1.9 x 102

(mos)

354 388 B2

Time in

Final population'

storagea

Agrobacterium radiobacter pv. radiobacter 84 A. tumefaciens

Dry film

21d

(CUm)(mos) 104 104 103 105

103 104

105 105

o

(CFU/mg)

(F/g

4d

0

16d

0

4d

0

id 2d id

0

0

27 27 38 38 38 18

6.1 x 103 io3 2.3 x 102 9.3 x 105 7.0 x 105 3.5 x 105 4.7 x 104

32 32 32 18

7.9 x 5.0 x 6.3 x 9.0 x

38 38

9.8 x 105 5.1 x 105

38 38

2.7 x 104 8.4 x 104

38 38

3.9 x 105 4.7 x 105

38 38

6.2 x 104 2.7x103

0

103 102 104 102

P. putida 38 B4 7.4 x 106 38 9.8 x 103 SH5 38 9.8 x 105 38 2.9 x 104 642 38 2.7 x 105 38 1.8 x 103 P. solanacearum 26 1.9 x 103 6d 0 P. syringae 11 pv. papulans T-1 3.4 x 105 11 NDe 38 2.5 x 103 pv. phaseolicola 279 38 1.9 x 103 30 1.7 x 102 pv. phaseolicola HB31 30 50 pv. phaseolicola HB36 38 2.8 x 103 38 3.4 x 102 pv. savastanoi 29 4.8 x 104 29 7.0 x 102 pv. syringae Lil-1 38 3.4 x 105 38 3.6 x 102 pv. tomato 38 2.9 x 102 38 1.0 x 102 P. viridaflava PC-1 7 7.0 x 105 7 ND Serratia marcescens 38 4.7 x 105 38 8.0 x 103 Xanthomonas campestris pv. begoniae 22 2.7 x 105 22 5.0 x 102 pv. campestris 855 38 3.5 x 104 1.9 x 10 38 pv. incanae 22 9.0 x 105 22 3.9 x 102 pv. juglandis 38 4.3 x 104 38 3.7 x 104 pv. malvacearum 874 38 4.0 x 104 38 3.0 x 103 pv. phaseoli 882 26 3.7 x 103 26 1.9 x 103 pv. vesicatoria 891 26 9.8 x 103 26 2.3 x 103 a Time of storage varies since some strains were introduced into the study at later times. b Initial populations for all strains were adjusted to approximately 10' CFU/ml of MC. ' Initial populations for all strains were adjusted to approximately 10' CFU/mg of MC (dry film weight) before drying and storage. d Viable populations could not be detected after this period. 'ND, not done.

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BACTERIAL PRESERVATION IN METHYLCELLULOSE

875

TABLE 2. Comparison of differential survival ability of E. chrysanthemi strains in frozen and film MC' CFU/mg of dry film MC after:

CFU/ml of frozen (-14°C) MC after:

Strain 4 days

30 days

90 days

CYC-P 107 108

2 x 105 0 2 x 102

1.7 x 105

8.9 x l04

111

1 X 102

0 0

113

1 X 102

1.5 x 102

0

2.3 x i03 1.7 x 102

0 0 0 1.2 x 102 1.9 X 102 2.5 x 103

114 116 120 123

0 0 0 0

136 137 138

7.2 x 2.6 x 3.5 x 2.8 x 1.2 x 5.5 X 0

Phil-i

i04 103 103 103 104 104

1 X

102

1.6 x 102 2.3 x 102 2 x 103

4 days

3.5 x

107

6.0 x 108 5.9 x 108 2.3 x 108 2.7 x 108 8.9 x107 8.0 x 107 2.6 x 107 2.0x108 4.3 x i07 6.9 x 107 6.0 x 107 3.5 x 107 3.0 x 108 9.9 X 107 1.2 x 108

90 days 2.4 x 107 1.3 x 102 1.9 X 102 4.3 x 102 0 0 0 0 0 5.9 X i04 1.7 x 102

6 mo 7.9 x 104 0 0 0

4.7 x 104 1.5 x 102

0

3.3 x 103 Phil-2 3.5 x 104 14A 7.9 x 105 14B 2.3 x 103 a Populations of initial bacteria-MC suspensions were adjusted to approximately 109 CFU/ml.

proved storage methods are based on a much more limited number of bacteria and a shorter time period, comparisons of populations at various time intervals clearly indicated the value of the improvements in reducing population declines. At this stage of development we recommend the following protocols for storing phytopathogenic bacteria. (i) Frozen storage. Bacterial strains are cultured on a complex medium, such as potato dextrose peptone or yeast dextrose calcium carbonate peptone, and then washed from medium in 0.1 M MgSO4. The bacteria are suspended in 1.0 MC containing 0.1 M MgSO4 and frozen at -14°C. Resuspensions from frozen storage are made in 0.1 M MgSO4. (ii) Dry film. Bacterial suspensions are prepared as for frozen storage. However, MgSO4 should not be added to the 1.0% MC suspension because the osmotic potential is too great upon drying for adequate bacterial survival (unpublished data). Resuspensions from dry-film storage are made in 0.1 M MgSO4. Storage of fims at 4°C in a desiccated environment may increase and extend the period of bacterial survival (un-

published data). Storage in frozen MC. Of 44 bacterial cultures, 41 were recovered from storage in frozen MC over periods ranging from 7 to 38 months (Table 1). These periods, however, do not represent finite periods of survival but were sampling times selected to coincide with the time of this study. The populations of bacteria after 38 months of storage ranged from 2.3 x 102 to 9.8 x 105 CFU/ml and varied depending upon species and strain. Strains of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica,

1.8 x 104 7.8 x 104 6.3 x 105 2.1 x 104

and Erwinia chrysanthemi did not survive longterm storage in frozen MC by the original procedures. Revised procedures that increased survival of these bacteria in short-term tests are presented above. S. marcescens, E. carotovora subsp. carotovora, X. campestris pv. begoniae, P. syringae pv. syringae, and P. fluorescens survived periodic freezing, thawing, and refreezing in individual vials of 1.0% MC. Although populations declined from initial populations of 5 x 10' CFU/ ml to final populations of 105 to 106 CFU/ml between isolation intervals, sufficient numbers survived for adequate recovery. E. carotovora subsp. carotovora did not survive successive freezings as well as other strains tested, declining to 7 X 103 CFU/ml after the fourth freezing interval. Storage in dry-film MC. Of 44 bacterial strains, 37 were susccessfully stored in dry-film MC, at ambient temperatures (23°C), for periods ranging from 18 to 38 months (Table 1). A drying temperature of 10'C was most conducive to survival for all strains tested. Many Pseudomonas, Xanthomonas, Corynebacterium, and S. marcescens isolates survived equally well at the more rapid drying temperatures, 24 to 32°C. Strains of E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. chrysanthemi, and Pseudomonas solanacearum survived for periods ranging from 1 to 16 months at low populations. Strains of E. chrysanthemi differed markedly in their ability to survive storage in frozen and dry-film MC (Table 2). Seven of 17 frozen strains were viable after 6 months of storage at -14°C,

876

APPL. ENVIRON. MICROBIOL.

SUSLOW AND SCHROTH

and only 4 of 17 survived 90 days in dry film. E. chrysanthemi strains 14A, Phil-1, Phil-2, and Cyc-P were viable and maintained pathogenicity characteristics in storage. All isolates tested maintained pathogenicity for their period of viability. Effect of growth media on bacterial survival. Of the media tested, bacteria survived better in MC when culture on yeast dextrose calcium carbonate peptone and potato dextrose peptone than on KB or nutrient agar (Table 3). Survival and recovery were greatest for Erwinia spp. when cultured on potato dextrose peptone and Agrobacterium spp. when cultured on yeast dextrose calcium carbonate peptone. None of the bacteria tested survived 8 months of storage in dry fim when cultured on nutrient agar. Effect of MgSO4 on increasing bacterial recovery from MC. The addition of Mg2" ions to the bacteria-MC suspension significantly increased the survival and recovery of P. phaseolicola HB36 but not of A. radiobacter 84. Recovery of P. phaseolicola HB36 from frozen MC was increased 230% with the addition of 0.1 M MgSO4 to the freezing menstruum. Average populations per vial after 1 month were 2.7 x 106 CFU/ml and 8.9 x 106 CFU/ml for MC and MC + MgSO4 and 2.0 x 106 CFU/ml in MC alone. Colony formation, however, on solid KB medium was observed to be faster with A. radiobacter 84 from MC + MgSO4.

DISCUSSION Storage of bacterial strains in frozen or dryfilm cellulose methyl ether (MC) was a simple, fast, and effective method for preservation with most strains tested for periods up to 38 months. The effective storage period of many strains presumably is greater than the 38-month test period, as indicated by the populations of viable bacteria. The populations of bacteria surviving storage varied among species and strains, with some having a short survival period. The effects of amino acids, polyhydric alcohols, dextrans, and sugars used for protection of bacterial cultures from dehydration have been noted (19). Of the sugars, the larger trisaccharides were. claimed to give the best protection from desiccation. MC, also a long-chain polymer, would appear to maintain an environment protective from complete dehydration. Strange and Cox (19) claimed that residual moistures of 0.5 to 1.5%, as would be achieved with lyophilization, are optimal for long-term bacterial survival. MC films had a residual moisture of 12.7% after initial drying, which could explain continual population decline with storage. Storage of films under desiccant conditions may be more conducive to maintaining the bacteria in a hypobiotic state for survival. Magnesium ions also are important in prolonging bacterial viability, especially with the

TABLE 3. Effect of media on bacterial survival in frozen and dry-fii.n MC Survival (CFU6)

Medium" Sampling . period

KB

NA

PDP

YDCP

E. carotovora subsp. carotovora

E. carotovora subsp.

atroseptica

Frozen (106)

Dry film (106)

Frozen (106)

0 1 wk 1 mo 8 mo

350 0.07 0.02 0.03

510 0.5 0.0

350 2.6 2.1 2.3

0 1 wk 1 mo 8 mo

260 0.4 0.0 0.0

0 1 wk 1 mo 8 mo

100 11.5 12.6 13.0

0 1 wk 1 mo 8 mo

1,300 2.5 3.7 2.5

1,000 2.4 0.5 0.0

290 2.6 0.09 0.09

790 32.0 0.26

100 10.0

1.1

11.9

410 1.3 0.0

700 6.2 6.2

11.2

Dry film (105) 560

A. radiobacter 84 Frozen

(106) 1,300

A. tumefaciens 388

Dry film

Frozen

(105)

(106)

510

0.5 0

24.0

75.0

1,000 17.0

19.0 6.9

60.0 8.3

23.0 8.0

510 3.5

300 2.8

320 48.9

260 3.5

Dry film (105)

350

41.9 2.0 2.5 190 0.6

0.001

2.2

2.5

1.7

0.002

0.0

2.1

0.0

1.7

0

1,100

600

370

39.0 0.5 0.6

12.0 18.0 7.5

86.0 40.0 28.8

750 0.8

1,600 55.0 67.0

1,400 86.9

800 9.0 12.1 15.0

1,900 79.0

390 82.0 41.0 24.5 1,200 48.0

0.001 45.0 42.0 8.3 6.2 0.4 51.0 15.0 41.0 18.8 aKB, King medium B; NA, nutrient agar; PDP, potato dextrose peptone agar; YDCP, yeast dextrose calcium carbonate peptone agar. b For frozen cultures, CFU per milliliter of liquid MC; for dry-film cultures, CFU per milligram of film.

BACTERIAL PRESERVATION IN METHYLCELLULOSE

VOL. 42, 1981

gram-negative bacterial (12, 15). Suspending bacterial cells in distilled water, as was the method of reisolation from storage early in our study, strips Mg2+ from the cell membrane, causing damage, and increases subsequent damage by both freezing and thawing. The addition of 0.1 M MgSO4 to the suspending medium increased the survival of P. syringae pv. phaseolicola in frozen MC. The survival of strains, such as E. carotovora subsp. carotovora and P. solanacearum, that were more sensitive to freezing in MC may be increased with the addition of Mg2+ into a complex growth medium. The advantages of the MC culture preservation technique are the simplicity of preparation compared to other techniques, the maintenance of pathogenicity and biological activity characteristics, and the versatility of storage and reisolation. These techniques also can be easily modified to suit a particular need or storage vehicle. For example, cultures have been shipped through the mail as strips of film, or have been coated onto glass slides and glass beads for shipping. MC storage appears especially useful as a short- and long-term culture preservation technique which complements lyophilization.

7. 8. 9.

10.

11. 12.

13. 14.

15.

16.

17.

LITERATURE CITED 1. Brockwell, J. 1962. Studies on seed pelleting as an aid to legume seed. Aust. J. Agric. Res. 13:638-649. 2. Clement, M. 1961. Effects of freezing, freeze-drying, and storage in the freeze-dried and frozen state on viability of Escherichia coli cells. Can J. Microbiol. 7:99-106. 3. Clement, M. 1964. A simple method of maintaining stock culture by low-temperature storage. Can J. Microbiol. 10:613-615. 4. Davidson, F., and H. Reuszer. 1978. Persistence of Rhizobiumjaponicum on soybean seed coat under controlled temperature and humidity. Appl. Environ. Microbiol. 35:94-96. 5. Dommergues, Y., H. Diem, and C. Divies. 1971. Polyacrylamide-entrapped Rhizobium as an inoculant for legumes. Appl. Environ. Microbiol. 37:779-781. 6. Gilmour, M. N., G. Turner, R. G. Berman, and A. K.

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Krenzer. 1978. Compact liquid nitrogen storage system yielding high recoveries of gram-negative anaerobes. Appl Environ. Microbiol. 35:84-88. Grivell, A., and J. Jackson. 1969. Microbial culture preservation with silica gel. J. Gen. Microbiol. 58:423435. Heckley, R. 1961. Preservation of bacteria by lyophilization. Adv. Appl. Microbiol. 3:1. Howard, D. H. 1959. The preservation of bacteria by freezing in glycerol broth. J. Bacteriol. 71:625. Klement, Z., G. L. Farkas, and L. Lovrekovich. 1944. Hypersensitive reaction induced by phytopathogenic bacteria in the tobacco leaf. Phytopathology 54:474477. Lange, J., and W. Boyd. 1968. Preservation of fungal spores by drying on porcelain beads. Phytopathology 58:1711-1712. Macleod, R., and P. Calcott. 1976. Cold shock and freezing damage to microbes, p. 81-109 In T. Gray and J. Postgate (ed.), The survival of vegetative microbes. Cambridge University Press, Cambridge, U.K. Moore, L. W., and R. V. Carlson. 1975. Liquid nitrogen storage of phytopathogenic bacteria. Phytopathology 65:246-250. New, P. B., and A. Kerr. 1972. Biological control of crown gall: field measurements and glasshouse experiments. J. Appl. Bateriol. 35:279-287. Postgate, J. 1976. Death in macrobes and microbes, p. 118: In T. Gray and J. Postgate (ed.), The survival of vegetative microbes. Cambridge University Press, Cambridge, U.K. Quadling, C. 1960. Preservation of Xanthomonas by freezing in glycerol broth. Can. J. Microbiol. 6:475-477. Sleesman, J., and C. Leben. 1976. Bacterial desiccation: effects of temperature, relative humidity, and culture age on survival. Phytopathology 66:1334-1338. Sleesman, J., and C. Leben. 1978. Preserving phytopathogenic bacteria at -70°C or with silica gel. Plant Dis. Rep. 62:910-913. Strange, R., and C. Cox. 1976. Survival of dried and airborne bacteria, p. 111-154. In T. Gray and J. Postgate (ed.), The survival of vegetative microbes. Cambridge University Press, Cambridge, U.K. Suslow, T. V., and M. N. Schroth. 1978. Bacterial culture preservation in methyl cellulose. Phytopathol. News 12:136. Trollope, D. 1975. The preservation of bacteria and fungi on anhydrous silica gel: an assessment of survival over four years. J. Appl. Bacteriol. 38:115-120. Vidaver, A. 1977. Maintenance of viability and virulence of C'orvnebacterium nebraskense. Phytopathology 67: 825-827.

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