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Reviving H20 suspended mycelium


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#21 whitethumb

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Posted 01 January 2017 - 06:53 PM

how did i miss this thread??? nice write up you have here.

i take the tip of my needle and scrape the surface of the agar plate. then i squirt sterilized water on the surface then suck it back up into the syringe.
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#22 Needles

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Posted 01 January 2017 - 10:31 PM

how did i miss this thread??? nice write up you have here.
i take the tip of my needle and scrape the surface of the agar plate. then i squirt sterilized water on the surface then suck it back up into the syringe.


That sounds like a awesome technique whitethumb..... You could cap your syringe and save your culture for years that way. Then all you would have to do is noc a jar of grain when you are ready......

I have also found going back to agar with blended mycelium can take weeks before the mycelium recovers...........
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#23 GLP

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Posted 02 January 2017 - 07:51 AM

Thanks for the link Needles, I found several others but missed that one. Will be looking for a used blender on my travels over the next weeks, I have a few agar plate arriving shortly that I would like to try this on. Thank you again. :tinfoil:


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#24 Tenderfoot

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Posted 02 January 2017 - 04:44 PM

Needles,

 

I hope you do not mind me sharing this in your thread, but it seems relevant!  ( borrowed from Shroomery)

 

 

Revival of saprotrophic and mycorrhizal basidiomycete cultures after 20 years in cold storage in sterile water.

 

Subject: Water, Distilled (Properties)

Fungi (Storage)

Cold storage (Methods)

Cultures (Biology) (Management)

Author: Richter, Dana L.

Pub Date: 08/01/2008

Publication: Name: Canadian Journal of Microbiology Publisher: NRC Research Press Audience: Academic Format: Magazine/Journal Subject: Biological sciences Copyright: COPYRIGHT 2008 NRC Research Press ISSN: 0008-4166

Issue: Date: August, 2008 Source Volume: 54 Source Issue: 8

Topic: Event Code: 200 Management dynamics Computer Subject: Company business management

Product: Product Code: 3841333 Culture-Based Tests NAICS Code: 339112 Surgical and Medical Instrument Manufacturing SIC Code: 3841 Surgical and medical instruments

Geographic: Geographic Scope: United States Geographic Code: 1USA United States

 

Abstract: Vegetatively colonized agar cores of 69 basidiomycete fungus isolates (48 species in 30 genera and 17 families) were stored at 5 [degrees]C in tubes of sterile distilled water without manipulation for 20 years. These were represented by 34 isolates of saprotrophic fungi (29 species in 19 genera) and 35 isolates of mycorrhizal fungi (19 species in 11 genera). Viability was evaluated based on revived growth on agar media at room temperature. Fifty-seven of the 69 isolates (82.6%) grew vigorously when revived after storage for 20 years; of the 34 saprotrophic fungus isolates, 30 revived (88.2%); of the 35 mycorrhizal fungus isolates, 27 revived (77.1%). Thirteen isolates of Laccaria were all viable after 20 years, indicating cold storage in sterile water to be a good method for maintaining this important genus of mycorrhizal fungi. In general, however, mycorrhizal fungus species demonstrated lower viability than saprotrophic fungi.

Key words: culture maintenance, culture viability, fungal preservation, long-term storage, mycorrhizal fungi, saprotrophic fungi, vegetative cultures.

Resume : Des echantillons d'agar colonises de facon vegetative comprenant 69 isolats de champignons basidiomycetes (48 especes divisees en 30 genres et 17 familles) ont ete entreposes a 5[degrees]C dans des tubes d'eau sterile distillee sans manipulation pendant 20 ans. Ceux-ci etaient representes par 34 isolats de champignons saprotrophes (29 especes divisees en 19 genres) et 35 isolats de mycorrhizes (19 especes divisees en 11 genres). La viabilite a ete evaluee par une reprise de la croissance sur agar a la temperature de la piece. Cinquante-sept des 69 isolats (82.6 %) poussaient vigoureusement lorsque ravives apres un entreposage de 20 ans; des 34 isolats de champignons saprotrophes, 30 ont ete ravives (88.2 %); des 35 isolats de mycorrhizes, 27 ont ete ravives (77.1 %). Les 13 isolats de Laccaria etaient tous vivants apres 20 ans, indiquant que l'entreposage au froid dans l'eau sterile est une bonne methode de conservation de ce genre important de mycorrhizes. En general, cependant, les especes de mycorrhizes demontraient une plus faible viabilite que les champignons saprotrophes.

Mots-cles : maintien des cultures, viabilite des cultures, preservation des champignons, entreposage a long-terme, mycorrhizes, champignons saprotrophes, cultures vegetatives.

[Traduit par la Redaction]

Introduction

Long-term storage of fungus cultures to ensure isolate stability is an important part of any mycology research laboratory. Interest in preserving microbial genomic diversity has also led to interest in maintaining cultures of fungi (Smith et al. 1994). However, maintaining genetic stability can be problematic. For example, characteristics of fungi such as pathogenicity, virulence, and growth rate are known to change over time when mycelium is continually sub-cultured on agar (Marx et al. 1984; Hung and Molina 1986; Richter et al. 2004). Advantages and disadvantages of the various methods of maintaining fungus cultures over long periods of time for research are thoroughly discussed by Smith and Onions (1983). A simple method of maintaining fungus cultures that has been successful in several laboratories in recent years is storage under sterile water in normal refrigeration (Marx and Daniel 1976; Ellis 1979; Richter and Bruhn 1989; Johnson and Martin 1992; Burdsall and Dorworth 1994; Smith et al. 1994).

Richter and Bruhn (1989) stored 135 basidiomycete fungus isolates, represented by 83 species in 38 genera, in sterile cold water (5 [degrees]C); of those that were revived after 8-48 months, 35 out of a total of 37 isolates of saprotrophic fungi were viable (95%), while only 53 out of a total of 98 isolates of mycorrhizal fungi were viable (54%). The original sterile water tubes containing cores of the isolates that were revived and viable were placed back in cold storage and left unmanipulated for 20 years. Of the original 135 isolates (Richter and Bruhn 1989), 88 isolates remained. However, due to difficulties inherent in storage conditions over such an extended period, several tubes dried or became contaminated. Thus, for this study, 69 isolates (34 saprotrophic fungi, 35 mycorrhizal fungi) were taken out of storage and attempts made to revive them after 20 years of storage in sterile cold water.

Materials and methods

Fungus isolates were originally obtained from basidiome tissue. Saprotrophic fungi were isolated on potato dextrose agar (Difco) or 2% malt extract agar (Difco), while mycorrhizal fungi were isolated on Modified Melin Norkrans agar (Marx 1969). Isolates were placed into sterile cold water storage 3-10 months after the time of isolation following the methods of Marx and Daniel (1976) (see Richter and Bruhn 1989). In this process, a sterile cork borer (8 mm diameter) was used to cut colonized agar cores from the margin of actively growing cultures in Petri dishes; agar was approximately 5 mm thick. Eight to 12 cores of each fungus were placed in 20 mL of sterile distilled water in a 20 mm x 150 mm glass culture tube; screw tops were placed on tightly and sealed with several wraps of Parafilm to minimize the chance of contamination or evaporation.

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For revival, 3 cores of each isolate were taken out of the sterile water tube and placed mycelium-side down on the surface of fresh agar media in Petri plates containing the medium on which they were originally grown and stored. Plates were incubated at room temperature and monitored weekly for up to 6 weeks for evidence of growth. Growth was compared with an actively growing culture of the fungus species freshly transferred from an agar slant. After examination, isolates were rated either viable or nonviable based on resumed growth or lack of growth, respectively.

Results and discussion

Results are shown in Table 1. For comparison, isolates are grouped in families as in the earlier paper that reported these isolates (Richter and Bruhn 1989). However, family affiliations have changed for some of the fungi and the modern classification is used here (Kirk et al. 2001).

The 69 fungus isolates for which revival was attempted were represented by 48 species, in 30 genera, in 17 families; 34 of these isolates were saprotrophic fungi (29 species, in 19 genera), and 35 isolates were mycorrhizal fungi (19 species, in 11 genera).

Of the 69 isolates, 57 (82.6%) grew vigorously when revived after 20 years of storage in sterile cold water; 30 of the 34 saprotrophic fungus isolates revived (88.2%), and 27 of the 35 mycorrhizal fungus isolates revived (77.1%).

For the viable isolates, all 3 of the cores that were retrieved from sterile water for each fungus grew new mycelium, except for 2 isolates of Laccaria laccata, in which case only one of the cores was viable for each (Table 1).

Remarkably, all 13 isolates of the mycorrhizal Laccaria bicolor and Laccaria laccata isolates survived after 20 years. However, only 4 of the 7 mycorrhizal Suillus species isolates survived. A large group of principally saprotrophic fungi, the Tricholomataceae, were generally viable, with 14 of 17 isolates reviving. Within this family, only one of 3 isolates of the genus Tricholoma (mycorrhizal) was not viable. Good viability was also exhibited in the saprotrophic Marasmiaceae (all 5 isolates viable) and the Pleurotaceae (all 3 isolates viable).

Of the 6 isolates of gasteromycetes, only one was not viable, this being the mycorrhizal species Scleroderma citrinum. This is not unexpected, since in the previous study (Richter and Bruhn 1989), 22 isolates in the genus Scleroderma exhibited very low survivability even after just 1 year of storage in sterile cold water.

The fungi in this study were essentially a select group of isolates in that these were isolates that had been stored, revived, and survived up to 4 years in cold water storage from the previous study (generally 2 years storage for most isolates) (Richter and Bruhn 1989). When the percentage of survival is calculated based on the original number of isolates (135) stored (minus 19 isolates not attempted in 2006 due to dryness and (or) contamination), this results in an overall survival rate after 20 years of 49.1% for all fungi. However, this percentage is lowered by the high number of mycorrhizal fungi in the original set. For the saprotrophic fungi alone, the survival rate from the original isolates after 20 years storage was 93.8%. In contrast, for the mycorrhizal fungi the survival rate after 20 years was only 32.1%. However, the percentage of survival for the mycorrhizal fungi is skewed downward due to the high number (22) of isolates of Scleroderma, which had a very low survival rate even after just 12 months of storage (Richter and Bruhn 1989). If isolates of Scleroderma are removed from the data set, the percentage of survival of mycorrhizal fungi after 20 years in sterile cold water becomes 43.5%.

Table 2 compares survival by genus after 2-4 years (Richter and Bruhn 1989) and 20 years of storage (this study), for those genera where 2 or more isolates were originally stored in sterile cold water. Genera of both mycorrhizal and saprotrophic fungi vary in their survivability after long-term storage in sterile cold water. For example, as mentioned above, the mycorrhizal genus Laccaria appears extremely well-suited for sterile cold-water storage. In contrast, the mycorrhizal genera Boletus, Lactarius, Paxillus, Scleroderma, and Thelephora are unsuited for this type of culture storage. Other mycorrhizal genera are intermediate in their survival success after long-term storage. In general, most isolates in genera of saprotrophic fungi survived after 20 years of storage in sterile cold water, with the exception of Clitocybe, of which 3 of 8 isolates failed to revive.

Burdsall and Dorworth (1994) also demonstrated a high rate of survivability (94%) of saprotrophic basidiomycete fungus cultures in sterile cold water for up to 7 years of storage. However, Johnson and Martin (1992), who stored their cultures of saprotrophic basidiomycete fungi at room temperature in sterile water for 10 years, reported only 26% survivability.

Smith et al. (1994), who stored cultures of mycorrhizal fungi for up to 20 months, demonstrated that temperature is a factor to be considered when storing cultures; in their study 95% of cultures revived at 18 [degrees]C, while at 4 [degrees]C only 78% of cultures revived. Marx and Daniel (1976), who stored mycorrhizal fungus cultures for up to 3 years in sterile cold water, showed that survival was 100% after 1 year but reduced to 95% and 64% after 2 and 3 years of storage, respectively. Based on this, although no other studies have reported revival of cultures of mycorrhizal fungi after 20 years of storage, 32.9% appears to be an expected rate of survival for this length of time.

It is of further interest that 21 of the isolates that were revived after 20 years in sterile cold water in this study did not survive after 8-12 years of transfer annually on agar slants (see Table 1). This was true for 11 of the 13 Laccaria isolates, further indicating sterile cold water to be a superior method for maintaining isolates of this genus of fungi. The mycorrhizal genus Suillus also had a high percentage of isolates that faired better stored in sterile cold water than by transferring annually on agar slants.

In conclusion, sterile cold water storage is a simple and effective method of long-term storage of basidiomycete fungus cultures; however, functional group and family must be taken into account when considering this method for use by laboratories. Overall, based on this study, this method of long-term storage is highly suitable for isolates of saprotrophic basidiomycete fungi, but this same generalization cannot be made for mycorrhizal fungi. Although sterile cold water storage appears to be a particularly good method to store isolates of the mycorrhizal genus Laccaria, it is not suitable for storing isolates of many other genera. Families and genera of mycorrhizal fungi exhibit a highly variable response to long-term storage in sterile cold water.

Acknowledgements

The former Institute of Wood Research at Michigan Technological University and the wood science program of Dr. Peter E. Laks allowed for long-term maintenance of these fungus cultures. Ms. Laura C. Kangas and Ms. Maureen L. Habarth are thanked for laboratory assistance and a careful reading of the manuscript.

Received 13 February 2008. Revision received 7 May 2008. Accepted 13 May 2008. Published on the NRC Research Press Web site at cjm.nrc.ca on 28 June 2008.

References

Burdsall, H.H., Jr., and Dorworth, E.B. 1994. Preserving cultures of wood-decaying Basidiomycotina using sterile distilled water in cryovials. Mycologia, 86: 275-280. doi:10.2307/3760650.

Ellis, J.J. 1979. Preserving fungus strains in sterile water. Mycologia, 71: 1072-1075. doi:10.2307/3759297.

Hung, L.L., and Molina, R. 1986. Temperature and time in storage influence the efficacy of selected isolates of fungi in commercially produced ectomycorrhizal inoculum. For. Sci. 32: 534-545.

Johnson, G.C., and Martin, A.K. 1992. Survival of wood-inhabiting fungi stored for 10 years in water and under oil. Can. J. Microbiol. 38: 861-864. doi:10.1139/m92-140.

Kirk, P.M., Cannon, P.F., David, J.C., and Stalpers, J.A. 2001. Ainsworth and Bisby's dictionary of the fungi. 9th ed. CABI, Oxfordshire, UK.

Marx, D.H. 1969. The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology, 59: 153-163.

Marx, D.H., and Daniel, W.J. 1976. Maintaining cultures of ectomycorrhizal and plant pathogenic fungi in sterile water cold storage. Can. J. Microbiol. 22: 338-341. PMID:1252993. doi:10. 1139/m76-051.

Marx, D.H., Cordell, C.E., Kenney, D.S., Mexal, J.G., Artman, J.D., Riffle, J.W., and Molina, R.J. 1984. Commercial vegetative inoculum of Pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on bare-root tree seedlings. For. Sci. Monogr. 25: 1-101.

Richter, D.L., and Bruhn, J.N. 1989. Revival of saprotrophic and mycorrhizal basidiomycete cultures from cold storage in sterile water. Can. J. Microbiol. 35: 1055-1060. doi:10.1139/m89-176.

Richter, D.L., Laks, P.E., Larsen, K.M., and Stephens, A.L. 2004. Comparison of isolates and strains within the brown rot fungus genus Gloeophyllum using the soil block decay method. For. Prod. J. 55: 72-75.

Smith, D., and Onions, A.H.S. 1983. The preservation and maintenance of living fungi. Commonwealth Mycology Institute, Kew.

Smith, J.E., McKay, D., and Molina, R. 1994. Survival of mycorrhizal fungal isolates stored in sterile water at tow temperatures and retrieved on solid and liquid nutrient media. Can. J. Microbiol. 40: 736-742. doi:10.1139/m94-117.

D.L. Richter. School of Forest Resources and Environmental Science, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA. ([email protected]).

Table 1. Survival of basidiomycete cultures in sterile cold water

after 20 years of storage.

Fungus species by family Isolate No. *

Agaricaceae

Leucoagaricus naucinus (Fr.) Singer DR-83

Macrolepiota procera (Scop.:Fr.) Singer DR-127

Bolbitaceae

Hebeloma crustuliniforme (Bull. DR-32

ex St. Am.) Quel.

Hebeloma sp. "A" DR-11

Hebeloma sp. "B" DR-110

Boletaceae

Boletus hosenae Smith & Thiers DR-28

Leccinum scabrum (Fr.) S. F. Gray DR-22

Hydangiaceae

Laccaria bicolor (Maire) Orton DR-64

Laccaria bicolor (Maire) Orton DR-72

Laccaria bicolor (Maire) Orton DR-91

Laccaria bicolor (Maire) Orton DR-100

Laccaria bicolor (Maire) Orton DR-112

Laccaria bicolor (Maire) Orton DR-141

Laccaria laccata (Scop.:Fr.) Berk. & Br. DR-5

Laccaria laccata (Scop.:Fr.) Berk. & Br. DR-95

Laccaria laccata (Scop.:Fr.) Berk. & Br. DR-102

Laccaria laccata (Scop.:Fr.) Berk. & Br. DR-113

Laccaria laccata (Scop.:Fr.) Berk. & Br. DR-115

Laccaria laccata (Scop.:Fr.) Berk. & Br. DR-133

Laccaria laccata (Scop.:Fr.) Berk. & Br. DR-137

 

Hygrophoropsidaceae

Hygrophoropsis aurantiaca (Wulfen:Fr.) Maire ATCC 60968

Hygrophoropsis aurantiaca (Wulfen:Fr.) Maire DR-66

 

Lycoperdaceae

Calvatia gigantea (Pers.) Lloyd DR-105

Lycoperdon muscorum Morgan DR-98

Lycoperdon perlatum Pers. DR-84

Marasmiaceae

Armillaria mellea sensu lato DR-52

Armillaria mellea sensu lato DR-86

Armillaria gallica Merxm. & Romagn. DR-140

Lentinula edodes (Berk.) Pegler DR-89

Oudemansiella radicata (Re1.:Fr.) Sing. DR-88

 

Paxillaceae

Paxillus involutus (Batsch:Fr.) Fr. DR-117

Phallaceae

Phallus impudicus (L.) Pers. DR-90

 

Pleurotaceae

Pleurotus ostreatus (Jacq.:Fr.) Kum. DR-85

Pleurotus ostreatus (Jacq.:Fr.) Kum. DR-93

Pleurotus ostreatus (Jacq.:Fr.) Kum. DR-153

Plutaceae

Amanita citrina (Schaeff) S. F. Gray DR-35

Amanita flavoconia Atk. DR-94

Amanita muscaria (L.:Fr.) Hooker DR-59

 

Rhizopogonaceae

Rhizopogon rubescens (Tul.) Tul. DR-128

Russulaceae

Lactarius rufus (Scop.:Fr.) Fr. DR-71

 

Sclerodermataceae

Scleroderma citrinum Pers. DR-134

Strophariaceae

Naematoloma sp. DR-145

Pholiota flammans (Fr.) Kum. DR-78

Pholiota sp. "A" DR-50

Pholiota sp. "B" DR-146

Suillaceae

Suillus luteus (Fr.) S. F. Gray DR-37

Suillus luteus (Fr.) S. F. Gray DR-82

Suillus luteus (Fr.) S. F. Gray DR-143

Suillus neoalbidipes Palm & Stewart DR-9

Suillus neoalbidipes Palm & Stewart DR-44

Suillus pictus (Pk.) Smith & Thiers DR-21

Suillus pictus (Pk.) Smith & Thiers DR-92

 

Tricholomataceae

Cantharellula umbonata (Gme1.:Fr.) Singer ATCC 62011

Clitocybe clavipes (Fr.) Kum. DR-38

Clitocybe dealbata (Sow.:Fr.) Gillet DR-33

Clitocybe geotropa (Bull. ex St. Am.) Kum. DR-139

Clitocybe gibba (Pers.:Fr.) Kum. DR-16

Clitocybe hydrogramma (Bull.:Fr.) Kum. DR-67

Clitocybe odora (Bull.:Fr.) Kum. DR-3

Clitocybe sp. DR-7

Collybia sp. DR-40

Hypsizygus tessulatus (Bull.:Fr.) Singer DR-129

Lepista glaucocana (Bres.) Singer DR-138

Lepista nuda (Bull.:Fr.) Cooke DR-147

Lyophyllum decastes (Fr. ex Fr.) Singer DR-87

Panellus stypticus (Bull.:Fr.) Karst. DR-106

Tricholoma populinum Lange DR-148

Tricholoma populinum Lange DR-149

Tricholoma resplendens (Fr.) Quel. DR-79

Fungus species by family Type of fungus

Agaricaceae

Leucoagaricus naucinus (Fr.) Singer Saprotrophic

Macrolepiota procera (Scop.:Fr.) Singer Saprotrophic

Bolbitaceae

Hebeloma crustuliniforme (Bull. Mycorrhizal

ex St. Am.) Quel.

Hebeloma sp. "A" Mycorrhizal

Hebeloma sp. "B" Mycorrhizal

Boletaceae

Boletus hosenae Smith & Thiers Mycorrhizal

Leccinum scabrum (Fr.) S. F. Gray Mycorrhizal

Hydangiaceae

Laccaria bicolor (Maire) Orton Mycorrhizal

Laccaria bicolor (Maire) Orton Mycorrhizal

Laccaria bicolor (Maire) Orton Mycorrhizal

Laccaria bicolor (Maire) Orton Mycorrhizal

Laccaria bicolor (Maire) Orton Mycorrhizal

Laccaria bicolor (Maire) Orton Mycorrhizal

Laccaria laccata (Scop.:Fr.) Berk. & Br. Mycorrhizal

Laccaria laccata (Scop.:Fr.) Berk. & Br. Mycorrhizal

Laccaria laccata (Scop.:Fr.) Berk. & Br. Mycorrhizal

Laccaria laccata (Scop.:Fr.) Berk. & Br. Mycorrhizal

Laccaria laccata (Scop.:Fr.) Berk. & Br. Mycorrhizal

Laccaria laccata (Scop.:Fr.) Berk. & Br. Mycorrhizal

Laccaria laccata (Scop.:Fr.) Berk. & Br. Mycorrhizal

 

Hygrophoropsidaceae

Hygrophoropsis aurantiaca (Wulfen:Fr.) Maire Saprotrophic

Hygrophoropsis aurantiaca (Wulfen:Fr.) Maire Saprotrophic

 

Lycoperdaceae

Calvatia gigantea (Pers.) Lloyd Saprotrophic

Lycoperdon muscorum Morgan Saprotrophic

Lycoperdon perlatum Pers. Saprotrophic

Marasmiaceae

Armillaria mellea sensu lato Saprotrophic

Armillaria mellea sensu lato Saprotrophic

Armillaria gallica Merxm. & Romagn. Saprotrophic

Lentinula edodes (Berk.) Pegler Saprotrophic

Oudemansiella radicata (Re1.:Fr.) Sing. Saprotrophic

 

Paxillaceae

Paxillus involutus (Batsch:Fr.) Fr. Mycorrhizal

Phallaceae

Phallus impudicus (L.) Pers. Saprotrophic

 

Pleurotaceae

Pleurotus ostreatus (Jacq.:Fr.) Kum. Saprotrophic

Pleurotus ostreatus (Jacq.:Fr.) Kum. Saprotrophic

Pleurotus ostreatus (Jacq.:Fr.) Kum. Saprotrophic

Plutaceae

Amanita citrina (Schaeff) S. F. Gray Mycorrhizal

Amanita flavoconia Atk. Mycorrhizal

Amanita muscaria (L.:Fr.) Hooker Mycorrhizal

 

Rhizopogonaceae

Rhizopogon rubescens (Tul.) Tul. Mycorrhizal

Russulaceae

Lactarius rufus (Scop.:Fr.) Fr. Mycorrhizal

 

Sclerodermataceae

Scleroderma citrinum Pers. Mycorrhizal

Strophariaceae

Naematoloma sp. Saprotrophic

Pholiota flammans (Fr.) Kum. Saprotrophic

Pholiota sp. "A" Saprotrophic

Pholiota sp. "B" Saprotrophic

Suillaceae

Suillus luteus (Fr.) S. F. Gray Mycorrhizal

Suillus luteus (Fr.) S. F. Gray Mycorrhizal

Suillus luteus (Fr.) S. F. Gray Mycorrhizal

Suillus neoalbidipes Palm & Stewart Mycorrhizal

Suillus neoalbidipes Palm & Stewart Mycorrhizal

Suillus pictus (Pk.) Smith & Thiers Mycorrhizal

Suillus pictus (Pk.) Smith & Thiers Mycorrhizal

 

Tricholomataceae

Cantharellula umbonata (Gme1.:Fr.) Singer Saprotrophic

Clitocybe clavipes (Fr.) Kum. Saprotrophic

Clitocybe dealbata (Sow.:Fr.) Gillet Saprotrophic

Clitocybe geotropa (Bull. ex St. Am.) Kum. Saprotrophic

Clitocybe gibba (Pers.:Fr.) Kum. Saprotrophic

Clitocybe hydrogramma (Bull.:Fr.) Kum. Saprotrophic

Clitocybe odora (Bull.:Fr.) Kum. Saprotrophic

Clitocybe sp. Saprotrophic

Collybia sp. Saprotrophic

Hypsizygus tessulatus (Bull.:Fr.) Singer Saprotrophic

Lepista glaucocana (Bres.) Singer Saprotrophic

Lepista nuda (Bull.:Fr.) Cooke Saprotrophic

Lyophyllum decastes (Fr. ex Fr.) Singer Saprotrophic

Panellus stypticus (Bull.:Fr.) Karst. Saprotrophic

Tricholoma populinum Lange Mycorrhizal

Tricholoma populinum Lange Mycorrhizal

Tricholoma resplendens (Fr.) Quel. Mycorrhizal

Fungus species by family Viability

([dagger])

 

Agaricaceae

Leucoagaricus naucinus (Fr.) Singer +

Macrolepiota procera (Scop.:Fr.) Singer -

 

Bolbitaceae

Hebeloma crustuliniforme (Bull. +

ex St. Am.) Quel.

Hebeloma sp. "A" +

Hebeloma sp. "B" -

 

Boletaceae

Boletus hosenae Smith & Thiers +

Leccinum scabrum (Fr.) S. F. Gray -

 

Hydangiaceae

Laccaria bicolor (Maire) Orton (+)

Laccaria bicolor (Maire) Orton (+)

Laccaria bicolor (Maire) Orton +

Laccaria bicolor (Maire) Orton (+)

Laccaria bicolor (Maire) Orton (+)

Laccaria bicolor (Maire) Orton (+)

Laccaria laccata (Scop.:Fr.) Berk. & Br. (+)

Laccaria laccata (Scop.:Fr.) Berk. & Br. (+)

Laccaria laccata (Scop.:Fr.) Berk. & Br. (+)

Laccaria laccata (Scop.:Fr.) Berk. & Br. (+1)

Laccaria laccata (Scop.:Fr.) Berk. & Br. (+)

Laccaria laccata (Scop.:Fr.) Berk. & Br. (+)

Laccaria laccata (Scop.:Fr.) Berk. & Br. +

Hygrophoropsidaceae

Hygrophoropsis aurantiaca (Wulfen:Fr.) Maire +

Hygrophoropsis aurantiaca (Wulfen:Fr.) Maire +

Lycoperdaceae

Calvatia gigantea (Pers.) Lloyd +

Lycoperdon muscorum Morgan +

Lycoperdon perlatum Pers. +

 

Marasmiaceae

Armillaria mellea sensu lato +

Armillaria mellea sensu lato +

Armillaria gallica Merxm. & Romagn. +

Lentinula edodes (Berk.) Pegler +

Oudemansiella radicata (Re1.:Fr.) Sing. +

Paxillaceae

Paxillus involutus (Batsch:Fr.) Fr. -

 

Phallaceae

Phallus impudicus (L.) Pers. +

Pleurotaceae

Pleurotus ostreatus (Jacq.:Fr.) Kum. +

Pleurotus ostreatus (Jacq.:Fr.) Kum. +

Pleurotus ostreatus (Jacq.:Fr.) Kum. +

 

Plutaceae

Amanita citrina (Schaeff) S. F. Gray +

Amanita flavoconia Atk. +

Amanita muscaria (L.:Fr.) Hooker (+)

Rhizopogonaceae

Rhizopogon rubescens (Tul.) Tul. +

 

Russulaceae

Lactarius rufus (Scop.:Fr.) Fr. +

Sclerodermataceae

Scleroderma citrinum Pers. -

 

Strophariaceae

Naematoloma sp. +

Pholiota flammans (Fr.) Kum. -

Pholiota sp. "A" +

Pholiota sp. "B" +

 

Suillaceae

Suillus luteus (Fr.) S. F. Gray (+)

Suillus luteus (Fr.) S. F. Gray (+)

Suillus luteus (Fr.) S. F. Gray -

Suillus neoalbidipes Palm & Stewart (+)

Suillus neoalbidipes Palm & Stewart +

Suillus pictus (Pk.) Smith & Thiers -

Suillus pictus (Pk.) Smith & Thiers (-)

Tricholomataceae

Cantharellula umbonata (Gme1.:Fr.) Singer +

Clitocybe clavipes (Fr.) Kum. (+)

Clitocybe dealbata (Sow.:Fr.) Gillet +

Clitocybe geotropa (Bull. ex St. Am.) Kum. -

Clitocybe gibba (Pers.:Fr.) Kum. (+)

Clitocybe hydrogramma (Bull.:Fr.) Kum. +

Clitocybe odora (Bull.:Fr.) Kum. -

Clitocybe sp. +

Collybia sp. +

Hypsizygus tessulatus (Bull.:Fr.) Singer (+)

Lepista glaucocana (Bres.) Singer +

Lepista nuda (Bull.:Fr.) Cooke +

Lyophyllum decastes (Fr. ex Fr.) Singer (+)

Panellus stypticus (Bull.:Fr.) Karst. +

Tricholoma populinum Lange -

Tricholoma populinum Lange +

Tricholoma resplendens (Fr.) Quel. (+)

 

* DR, Collection of Dana L. Richter; ATCC, American Type Culture

Collection, Beltsville, Maryland.

([dagger]) Three agar cores were plated for each fungus. +, indicates

3 cores were viable; +1, indicates only one of 3 cores was

viable; -, indicates 3 cores were not viable. Data in parentheses

indicate that the culture did not survive after 8-12 years on

agar slants transferred annually.

Table 2. Summary of survival by genus of basidiomycete cultures in

sterile cold water after 2-4 and 20 years of storage in sterile cold

water (only genera with 2 or more original isolates stored are

shown).

 

Total no.

Fungus genus Type of fungus of isolates

Amanita Mycorrhizal 9

Armillaria Saprotrophic 3

Boletus Mycorrhizal 9

Clitocybe Saprotrophic 8

Hebeloma Mycorrhizal 4

Hygrophoropsis Saprotrophic 2

Laccaria Mycorrhizal 15

Lactarius Mycorrhizal 9

Leccinum Mycorrhizal 2

Lepista Saprotrophic 2

Lycoperdon Saprotrophic 2

Paxillus Mycorrhizal 3

Pholiota Saprotrophic 3

Pleurotus Saprotrophic 3

Scleroderma Mycorrhizal 22

Suillus Mycorrhizal 13

Thelephora Mycorrhizal 3

Tricholoma Mycorrhizal 4

 

No. of isolates surviving

after:

Fungus genus 2-4 years * 20 years

Amanita 3 3

Armillaria 3 3

Boletus 1 1

Clitocybe 7 5

Hebeloma 3 2

Hygrophoropsis 2 2

Laccaria 13 13

Lactarius 1 1

Leccinum 1 1

Lepista 2 2

Lycoperdon 2 2

Paxillus 1 0

Pholiota 3 2

Pleurotus 3 3

Scleroderma 1 0

Suillus 7 4

Thelephora 0 -

Tricholoma 3 2

 

* Data are from Richter and Bruhn (1989).

Gale Copyright: Copyright 2008 Gale, Cengage Learning. All rights reserved.


Edited by Tenderfoot, 02 January 2017 - 04:45 PM.

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#25 Tenderfoot

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Posted 02 January 2017 - 04:57 PM

Needles, again, my apologies if cluttering your thread!

 

https://www.shroomer...ter-in-cryovial


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#26 Needles

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Posted 02 January 2017 - 08:03 PM

Needles, again, my apologies if cluttering your thread!
 
https://www.shroomer...ter-in-cryovial

No apologies needed... I enjoy reading scientific research reports like that. It is good reading and encourages me to continue with my own research and experiments. I found it interesting how they used agar cores and not mycelium alone. It also lets us know that sterile water storage is not 100% foolproof. Their results showed a high percentage of recovery but not every culture survives.
They also use agar as a medium for recovery. I have found that it can take up to five weeks for blended mycelium to recover on agar.

Thanks for sharing the report, it is good info........
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#27 OysterFarmer

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Posted 05 January 2017 - 06:03 PM

I would think some species would take to this more than others.  Some I think just have a life cycle and then that's it for them.

 

Also why not just take a spore print?  Wouldn't that be easier?


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#28 Tenderfoot

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Posted 05 January 2017 - 08:27 PM

These will "outlive" prints, hypothetically!

 

 

 

Also why not just take a spore print?  Wouldn't that be easier?


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#29 Needles

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Posted 05 January 2017 - 09:41 PM

I would think some species would take to this more than others.  Some I think just have a life cycle and then that's it for them.
 
Also why not just take a spore print?  Wouldn't that be easier?

Some species can take cold temps and some like it warm. I am working on and experimenting with different methods of preserving mycelium.
Once you have a isolate that is adapt to your growing methods then saving that culture is essential to keeping that strain living.
A spore print can be a alternative method of keeping your mushroom. Although it would not have the characteristics of your isolated culture.
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#30 OysterFarmer

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Posted 07 January 2017 - 03:52 PM

 

These will "outlive" prints, hypothetically!

 

 

 

Also why not just take a spore print?  Wouldn't that be easier?

 

Dry prints or spore in lc?  I know a friend that brought me an old syringe as she'd screwed up the others.  It was about a year old and did dick.  It was from of the bigger spore vendors in the U.S. so I'm pretty sure it was storage issues.


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#31 MikeORhizal

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Posted 20 January 2017 - 05:33 AM

Wow great set up. I am currently trying to collect all my own lab equip. Where do you get those jars and tops, or do you make them yourself? How do you aerate your liquid cultures, on a shaker? I have made liquid cultures of several edible (Plurotus djamor, Plurotus ferulae, Shitake, Lentinula edodes etc.) and Ganoderma (resinaceum, australe, lucidum), Grifola frondosa, Laetiporus sulphurus, and also Trametes versi. but rather than sterile water media these were on a simple malt broth in Erlenmeyer flasks, sealed with foam bungs, and incubated at 25°C on a shaker for ~ 2 months. The lingninolytic spp. almost fully colonated the liquid media and produced EPS and solidified the media into a jelly like substance. Then I blended this media in and LFC and used your methods as above to innoculate several jars. I noticed you said air wasn't an issue, in some spp. they benefit from gaseous exchange and gentle agitation :) Such a great setup looks like a pro lab. Congrats.

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#32 Needles

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Posted 20 January 2017 - 07:35 PM

Wow great set up. I am currently trying to collect all my own lab equip. Where do you get those jars and tops, or do you make them yourself? How do you aerate your liquid cultures, on a shaker? I have made liquid cultures of several edible (Plurotus djamor, Plurotus ferulae, Shitake, Lentinula edodes etc.) and Ganoderma (resinaceum, australe, lucidum), Grifola frondosa, Laetiporus sulphurus, and also Trametes versi. but rather than sterile water media these were on a simple malt broth in Erlenmeyer flasks, sealed with foam bungs, and incubated at 25°C on a shaker for ~ 2 months. The lingninolytic spp. almost fully colonated the liquid media and produced EPS and solidified the media into a jelly like substance. Then I blended this media in and LFC and used your methods as above to innoculate several jars. I noticed you said air wasn't an issue, in some spp. they benefit from gaseous exchange and gentle agitation :) Such a great setup looks like a pro lab. Congrats.
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The lids are made from Waring blenders that I have found at thrift shops or in people's garage sales. I like your tech with using malt as a broth to expand your mycelium. It's interesting how you would take months to let it grow. I use magnetic stirrers with malt broth but for only a few days.
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#33 Arathu

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Posted 26 January 2017 - 07:18 PM

Wow....killer thread!

 

A


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#34 Needles

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Posted 25 March 2017 - 08:39 PM

All but two cultures were revived and put back on agar. From those cultures fresh jars of grain spawn and liquid innoculent were started and stored. All together over twenty filter patch bags were filled with 1500 grams of sawdust substrate and sterilized. So hopefully I get a few fruits this spring. It was finally nice and I was able to get to my out building and work in my stainless flow hood. Here's a picture of ten bags and jars ready to go.image.jpeg
Once they get a little more colonized I'll line them up for a group photo.
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#35 Arathu

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Posted 26 March 2017 - 03:01 AM

Awesome........I need the green jealous emoticon................

 

hahahahaha.............

 

Weather is breaking here too so things will start up in earnest very shortly.....

 

Can't wait to see more pics......... :biggrin:

 

A


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#36 Sicshroom

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Posted 28 March 2017 - 08:41 AM

This is beautiful

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#37 Needles

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Posted 03 May 2017 - 09:23 PM

I finely had a chance to get my sawdust blocks in a outside fruiting chamber. I will try to get a few photos of my set up in the next couple of days.
First I wanted to share a few photos from a bag of lions mane. As soon as I started to notice that it was starting to fruit in the bag I immediately thought of the invitro tech that @MLBjammer has posted and taught us about. I cut holes in the bags and filled them with prolly fiber. This is a awesome way to grow edible mushrooms, it is very clean and also keeps out the bugs.
image.jpeg image.jpeg

My sister in law offered to make us a soup from the lions mane, shiitake and Hypsyzigus ulmarius that we had fruiting
image.jpeg
It was a good start to this springs mushroom season....
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#38 Heirloom

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Posted 12 May 2017 - 10:44 PM

Nice write up , like your lab. Could you tell us about nutrient broth(s). Thanks
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#39 Needles

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Posted 13 May 2017 - 09:23 PM

Nice write up , like your lab. Could you tell us about nutrient broth(s). Thanks


Nutritional broth is simply barley malt and a pinch of gypsum. 400ml water to 40 grams of malt and a very little gypsum.
If agitated by magnetic stirring a culture can be grown and expanded to grains with a fast recovery time.
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#40 Needles

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Posted 13 May 2017 - 09:27 PM

Very excited to see Hypsizygus tessellatus pins on this block today. They should start to grow fairly fast and lots of them.

image.jpeg
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