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Preservation of yeasts and fungi by desiccation
PRESERVATION OF YEASTS AND FUNGI BY DESICCATION By MABEL RHODES National Collection of Type Cultures, Elstree, Herts (With 4 Text-figures)
Bacteria, in the form of dried cultures, are known to survive for long periods (Hammer, 1911; Rogers, 1914; Swift, 1921; Flosdorf & Mudd, 1938 and Stamp, 1947); morphological, physiological and antigenic characters, and virulence are retained in their original state, and degenerative changes associated with repeated subcultures are halted. A simple method of drying cultures was described to us by Prof. A. Sordelli, of Buenos Aires, and we have used it in the National Collection of Type Cultures since 1934. The viability of bacteria preserved by this method will be reported separately; the present paper gives the results obtained with cultures of fungi and yeasts up to the time the National Collection of Type Cultures stopped keeping these organisms. METHOD
Cultures for drying were grown on a solid medium; non-sporing organisms were dried as soon as good growth developed, and incubation of sporing genera was continued until spores were produced. A loopful of culture or a small piece of mycelium, where possible with spores, was rubbed up in a loopful of sterile horse serum on the inner wall of a plugged sterile test-tube about 8 x 60 mm. (Fig. 1). This tube was placed inside a slightly wider tube ( 10 x 150 mm.) at the bottom of which was a small amount of phosphorus pentoxide (Fig. 2). The outer tube was then heated above the level of the inner tube's wool plug and drawn out (Fig. 3). The outer tube was attached to a Cenco Hyvac pump for exhaustion and when the vacuum was satisfactory was sealed off at the constriction (Fig. 4). Dried cultures were stored at room temperature in the dark. To recover the organisms the outer tube was opened and the inner tube removed. About 1 ml. of liquid medium was pipetted into the small tube; suitable media were beer wort for most yeasts and many fungi, and glucose broth for the others. The tube containing medium was incubated at the appropriate temperature until growth appeared. RESULTS
After intervals varying from one month to thirteen years dried cultures of sixty-one different genera were tested for viability. Two or three weeks might elapse before growth was apparent and tubes were kept for at least a month before being discarded as sterile. The time lag observed with dried cultures is not unusual and may occur when transplants are made from 3-2
36
Transactions British Mycological Society
cultures on ordinary nutrient medium. Table I summarizes the viability in years but does not differentiate genera among the 405 isolates tested.
r.¥, f ,
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,
I
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Fig.
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Fig.
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.'
Fig. 3.
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:
Fig. 4.
There is little difference in the proportion of viable cultures at any time interval and for convenience in tabulating the results obtained with different genera, cultures opened within four years of drying are grouped
Preservation ofyeasts and fungi. Mabel Rhodes
37
together, as are cultures tested between five and eight years, and between nine and thirteen years. A list of the genera dried and the number of viable cultures are given in Table 2. Table
I.
Viabiliry inyears of dried cultures of fungi andyeasts
Years between drying and test Less than I 1-2 2-3
3-4 4-5
5-6
6-7 7-8 8-9 9- 10 10-11 II-12 12-1 3
13-14
No, tested
8 9
No. viable 6
8
22
22
II
5 5
7
16 9 II9
II
8 108
154
136
121
II2
87 4 7 10
74 4
5 9
Many genera were represented by a single isolate, but the value of the method can be seen in genera such as Saccharomyces, of which twenty-eight species were dried. On revival all cultures of Aspergillus spored well, and some genera spored better than the cultures of the same organisms that had been maintained for the same interval on the usual media. DISCUSSION
In the tests carried out several genera, such as Acladium, Ashbya and Beauveria, were represented by a single isolate and it cannot be assumed that other isolates or different species of these genera could be dried as successfully. However, the success achieved with many species of Cryptococcus, Endomycopsis, Monilia, Saccharomyces, and Trichophyton suggests that where a single isolate of a genus is capable of being dried other isolates or species of the same genus will behave in a similar way. From a much greater experience in the drying of bacteria we know that there are exceptions to a generalization of this sort. With the exception of Aleurisma, Phytophthora, Sabouraudites, Schwanniomyces, Sphaceloma and Trichosporium, in each of which only a single isolate was dried, all the genera tested were dried successfully. Over the period of tests 87'9% ofdried cultures were found to be viable; at the end of thirteen years the proportion of viable cultures was as great as the mean. Thus it appears likely that if a culture survives the drying process it will remain viable for a more or less indefinite period. SUMMARY
A simple technique, developed for preserving bacteria by drying, was tried on cultures of fungi and yeasts. Nearly 88% of cultures tested survived for varying periods up to thirteen years.
Table
2.
Viability of different genera kept in dried state Time (in years) between drying and test
-.....
A
No. of isolates
1-4 r-~
No. tested
No. viable
5-8 ~
No. tested
Acladium castellanii Aleurisma chartarum var. orientale Ashbyagossypii Aspergillus, 2 spp. 9 9 5 Atelosaccharomyces sp. I Beauoeria cylindrospora I Bodinia oiolacea 2 Botrytis allii I a Brettanomyces spp. 2 Candida, 6 spp. 7 Chalara mycoderma I Cladosporium mansonii I Coccidioides, 2 spp. 2 Cordyceps militaris I 21 Cryptococcus, I r s., 7 un. spp. Debaryomyces, 4 spp. 5 Endodermophyton, 2 spp. 4 Endomycopsis, 9 spp. 2 a 14 Epidermophyton, 3 spp. 3 Geotrichum, 8 spp, II 6 Glenospora, 4 spp. I Hansenia apiculata I Hanseniospora valbyensis Hansenula, 4 spp. 7 Hemispora stellata 2 Hormodendrum pedrosoi 2 Hyalodendron lignicola 3 16 Kloeckera, 15 spp, I Malassezia sp. 5 4Micromonospora, 3 spp. 3 2 Microsporum, 2 spp. Monilia, I9n., 5un. spp; 35 3 3 2 Mono:t:::::m, 2 spp. Myco ,4spP. 4Mycotorula, 7 spp. 13 I Nadsonia fuloescens Nematospora coryli 5 6 2 2 Oidium, 3 spp. Parasaccharomyces ashfordii a Phytophthora porn Pichia, 8 spp. 9 I Pitymsporum ooale 2 Prototheca, 2 spp. 10 Rhodotorula, 8 spp. I Sabouraudites felineus 0 Saccharomyces, 28 spp, 9 55 9 2 Saccharomycodes ludwigii Saccharomycopsis capsularis 2 Schi;;osaccharomyces, 3 spp, 2 44I Schwanniamyces occidentalis I Sphaceloma oiolae I Sporendonema epizoum I Sporobolomyces roseus Sporotrichum, 6 n., !Un. spp. 13 2 Torula, 22 n., 7 un, spp. 2 32 Ig Torulopsis, 17 spp. 5 5 12 Trichophyton, I I n., tun. spp. 2 3 I Trichosporon beigeli I Trichosporium tugosum 2
2
No. viable
9- 1 3 ~
No. tested
No. viable
2
a
5
5
2
2
9 I
9 I
3
3
I 2
I I I
I
18 3 3 14 2 8
15 3 I 12 2
16 2 2 6 3
15 2 2 6 2
4
7
7
7
4
2 2
2 2
I
I
4 4-
4 4-
5 5
2
2
3 3 2 6 10
2
6 2
2
5
2 2 22
422 3 10 I
3 10
9 5
9 4-
5
5
49 I
26 I
2 2 13 32 19 II I
I I
8 14-
species
21 I
I
33 2 2 13
33 2 2 13
2 2
2 2
7
7
2
2
28 2 2 2 I 2
28 2 2 2 0 0
I
49 0 26 0
2 2
13 32
9
7
9
6
16
2
2
3 I 0
7
6
I
I 2 14-
8 13
I I
2 13
Preservation of yeasts and fungi. Mabel Rhodes
39
Dr R. St John-Brooks started drying cultures by the method outlined in this paper and it is to his interest and encouragement that lowe my thanks. REFERENCES
FLOSDORF, E. W. & MUDD, S. (1938). An improved procedure and apparatus for preservation of sera, micro-organisms and other substances-the Cryochem-process. J. Immunol. 34, 469. HAMMER, B. W. (1911). A note on the vacuum desiccation of bacteria. J. med. Res. 24, 527. ROGERS, L. A. (1914). The preparation of dried cultures. J. infect. Dis. 14,100. STAMP, LORD (1947). The preservation of bacteria by drying. J. gen. Microbial. I, 251. SWIFT, H. F. (1921). Preservation of stock cultures by freezing and drying. J. expo Med. 33,69·
(Accepted for publication
IO
December 1948)
Bacteria, in the form of dried cultures, are known to survive for long periods (Hammer, 1911; Rogers, 1914; Swift, 1921; Flosdorf & Mudd, 1938 and Stamp, 1947); morphological, physiological and antigenic characters, and virulence are retained in their original state, and degenerative changes associated with repeated subcultures are halted. A simple method of drying cultures was described to us by Prof. A. Sordelli, of Buenos Aires, and we have used it in the National Collection of Type Cultures since 1934. The viability of bacteria preserved by this method will be reported separately; the present paper gives the results obtained with cultures of fungi and yeasts up to the time the National Collection of Type Cultures stopped keeping these organisms. METHOD
Cultures for drying were grown on a solid medium; non-sporing organisms were dried as soon as good growth developed, and incubation of sporing genera was continued until spores were produced. A loopful of culture or a small piece of mycelium, where possible with spores, was rubbed up in a loopful of sterile horse serum on the inner wall of a plugged sterile test-tube about 8 x 60 mm. (Fig. 1). This tube was placed inside a slightly wider tube ( 10 x 150 mm.) at the bottom of which was a small amount of phosphorus pentoxide (Fig. 2). The outer tube was then heated above the level of the inner tube's wool plug and drawn out (Fig. 3). The outer tube was attached to a Cenco Hyvac pump for exhaustion and when the vacuum was satisfactory was sealed off at the constriction (Fig. 4). Dried cultures were stored at room temperature in the dark. To recover the organisms the outer tube was opened and the inner tube removed. About 1 ml. of liquid medium was pipetted into the small tube; suitable media were beer wort for most yeasts and many fungi, and glucose broth for the others. The tube containing medium was incubated at the appropriate temperature until growth appeared. RESULTS
After intervals varying from one month to thirteen years dried cultures of sixty-one different genera were tested for viability. Two or three weeks might elapse before growth was apparent and tubes were kept for at least a month before being discarded as sterile. The time lag observed with dried cultures is not unusual and may occur when transplants are made from 3-2
36
Transactions British Mycological Society
cultures on ordinary nutrient medium. Table I summarizes the viability in years but does not differentiate genera among the 405 isolates tested.
r.¥, f ,
r· ..~ (
, ,.
,. ,
I
'
~\
\ 'I ,I'
,
I
I.
Fig.
I •
I
\ \ I
I
Fig.
\
2.
.'
Fig. 3.
\
:
Fig. 4.
There is little difference in the proportion of viable cultures at any time interval and for convenience in tabulating the results obtained with different genera, cultures opened within four years of drying are grouped
Preservation ofyeasts and fungi. Mabel Rhodes
37
together, as are cultures tested between five and eight years, and between nine and thirteen years. A list of the genera dried and the number of viable cultures are given in Table 2. Table
I.
Viabiliry inyears of dried cultures of fungi andyeasts
Years between drying and test Less than I 1-2 2-3
3-4 4-5
5-6
6-7 7-8 8-9 9- 10 10-11 II-12 12-1 3
13-14
No, tested
8 9
No. viable 6
8
22
22
II
5 5
7
16 9 II9
II
8 108
154
136
121
II2
87 4 7 10
74 4
5 9
Many genera were represented by a single isolate, but the value of the method can be seen in genera such as Saccharomyces, of which twenty-eight species were dried. On revival all cultures of Aspergillus spored well, and some genera spored better than the cultures of the same organisms that had been maintained for the same interval on the usual media. DISCUSSION
In the tests carried out several genera, such as Acladium, Ashbya and Beauveria, were represented by a single isolate and it cannot be assumed that other isolates or different species of these genera could be dried as successfully. However, the success achieved with many species of Cryptococcus, Endomycopsis, Monilia, Saccharomyces, and Trichophyton suggests that where a single isolate of a genus is capable of being dried other isolates or species of the same genus will behave in a similar way. From a much greater experience in the drying of bacteria we know that there are exceptions to a generalization of this sort. With the exception of Aleurisma, Phytophthora, Sabouraudites, Schwanniomyces, Sphaceloma and Trichosporium, in each of which only a single isolate was dried, all the genera tested were dried successfully. Over the period of tests 87'9% ofdried cultures were found to be viable; at the end of thirteen years the proportion of viable cultures was as great as the mean. Thus it appears likely that if a culture survives the drying process it will remain viable for a more or less indefinite period. SUMMARY
A simple technique, developed for preserving bacteria by drying, was tried on cultures of fungi and yeasts. Nearly 88% of cultures tested survived for varying periods up to thirteen years.
Table
2.
Viability of different genera kept in dried state Time (in years) between drying and test
-.....
A
No. of isolates
1-4 r-~
No. tested
No. viable
5-8 ~
No. tested
Acladium castellanii Aleurisma chartarum var. orientale Ashbyagossypii Aspergillus, 2 spp. 9 9 5 Atelosaccharomyces sp. I Beauoeria cylindrospora I Bodinia oiolacea 2 Botrytis allii I a Brettanomyces spp. 2 Candida, 6 spp. 7 Chalara mycoderma I Cladosporium mansonii I Coccidioides, 2 spp. 2 Cordyceps militaris I 21 Cryptococcus, I r s., 7 un. spp. Debaryomyces, 4 spp. 5 Endodermophyton, 2 spp. 4 Endomycopsis, 9 spp. 2 a 14 Epidermophyton, 3 spp. 3 Geotrichum, 8 spp, II 6 Glenospora, 4 spp. I Hansenia apiculata I Hanseniospora valbyensis Hansenula, 4 spp. 7 Hemispora stellata 2 Hormodendrum pedrosoi 2 Hyalodendron lignicola 3 16 Kloeckera, 15 spp, I Malassezia sp. 5 4Micromonospora, 3 spp. 3 2 Microsporum, 2 spp. Monilia, I9n., 5un. spp; 35 3 3 2 Mono:t:::::m, 2 spp. Myco ,4spP. 4Mycotorula, 7 spp. 13 I Nadsonia fuloescens Nematospora coryli 5 6 2 2 Oidium, 3 spp. Parasaccharomyces ashfordii a Phytophthora porn Pichia, 8 spp. 9 I Pitymsporum ooale 2 Prototheca, 2 spp. 10 Rhodotorula, 8 spp. I Sabouraudites felineus 0 Saccharomyces, 28 spp, 9 55 9 2 Saccharomycodes ludwigii Saccharomycopsis capsularis 2 Schi;;osaccharomyces, 3 spp, 2 44I Schwanniamyces occidentalis I Sphaceloma oiolae I Sporendonema epizoum I Sporobolomyces roseus Sporotrichum, 6 n., !Un. spp. 13 2 Torula, 22 n., 7 un, spp. 2 32 Ig Torulopsis, 17 spp. 5 5 12 Trichophyton, I I n., tun. spp. 2 3 I Trichosporon beigeli I Trichosporium tugosum 2
2
No. viable
9- 1 3 ~
No. tested
No. viable
2
a
5
5
2
2
9 I
9 I
3
3
I 2
I I I
I
18 3 3 14 2 8
15 3 I 12 2
16 2 2 6 3
15 2 2 6 2
4
7
7
7
4
2 2
2 2
I
I
4 4-
4 4-
5 5
2
2
3 3 2 6 10
2
6 2
2
5
2 2 22
422 3 10 I
3 10
9 5
9 4-
5
5
49 I
26 I
2 2 13 32 19 II I
I I
8 14-
species
21 I
I
33 2 2 13
33 2 2 13
2 2
2 2
7
7
2
2
28 2 2 2 I 2
28 2 2 2 0 0
I
49 0 26 0
2 2
13 32
9
7
9
6
16
2
2
3 I 0
7
6
I
I 2 14-
8 13
I I
2 13
Preservation of yeasts and fungi. Mabel Rhodes
39
Dr R. St John-Brooks started drying cultures by the method outlined in this paper and it is to his interest and encouragement that lowe my thanks. REFERENCES
FLOSDORF, E. W. & MUDD, S. (1938). An improved procedure and apparatus for preservation of sera, micro-organisms and other substances-the Cryochem-process. J. Immunol. 34, 469. HAMMER, B. W. (1911). A note on the vacuum desiccation of bacteria. J. med. Res. 24, 527. ROGERS, L. A. (1914). The preparation of dried cultures. J. infect. Dis. 14,100. STAMP, LORD (1947). The preservation of bacteria by drying. J. gen. Microbial. I, 251. SWIFT, H. F. (1921). Preservation of stock cultures by freezing and drying. J. expo Med. 33,69·
(Accepted for publication
IO
December 1948)