The #14 TEO was mailed out several days ago. It is the Issue that has Rodger Rabbit's article on how he brought the original 20 year old spore samples back.......a new novel method using Rattlesnake Venom!
I don't know if RR followed this lead or not, but here is some interesting
research about using snake venom as a hybridization agent.
We used purified snake venom from the Western Diamondback Rattlesnake (Crotalus atrox) for our hybridization techniques. The snake venom is added to the agar medium in quantities that alters the growth but does not prove toxic to the strain in question. This range of snake venom is from 10 mg to 30 mg per 300 ml of agar medium. The venom is not heat stable and must be added aseptically after sterilization of the medium. The agar used for this hybridization consisted of malt extract, activated carbon, minerals and humus – the carbon-rich ash residue from a coal burning industrial process. Other agars could probably be used as well. This just happens to be our production agar that we use everyday, and once we found that it also worked with the snake venom for hybridization.
Petri dishes of this R7 agar medium are inoculated with mycelium from two different strains of the Cordyceps genus. These different strains when inoculated together onto one petri dish will normally grow towards each other until they almost meet, at which point they form a zone of inhibition, where neither strain can grow. Eventually, one strain may prove stronger than the other and overgrow the plate, but they will remain genetically distinct; two different cultures residing in the same petri dish.
With the addition of a sufficient quantity of snake venom to the agar, we found that what happens is the two cultures grow towards each other until they meet and form their mutual zone of inhibition. This period of inhibition is short lived however, for in only about 2 or 3 hours the colonies each start sending out mycelial strands into this no-mans land, the zone of inhibition. These strands grow together and exchange nuclear material through their venom-weakened cell walls. They form a hybrid strain at this point of mutual contact. A new strain, one that is distinctly different from either of the parent strains. Within about 4 hours after first forming the zone of inhibition, the hybridization is complete and the colonies resume rapid growth towards each other. They become three colonies rather than the original two. There then exist in the same plate the original two colonies and a genetically distinct third…The Hybrid.
A section of the newly formed hybrid is carefully removed from the original zone of inhibition at the precise time that the colonies begin to fuse. That is during hour 3-4 after the initial meeting of the colonies. The hybrid is transferred to a new petri dish containing normal (non-snake venom) agar.
Our quick method of determining hybridization is to inoculate a new dish containing normal agar with all three strains, the original two and the suspected hybrid. If the hybridization has in fact taken place, these are now three distinct colonies, and will form a mutual three-way zone of inhibition. If hybridization has failed to occur, then the suspected hybrid will readily fuse with either one or the other of the original colonies. This proves that our suspected hybrid is not genetically distinct from the original and we start anew.
Once a hybrid is confirmed, it is tested for growth parameters. If it appears to be a vigorous and hardy grower on our substrate of choice, we grow out a quantity of mycelium, harvest it and analyze it for active ingredients. Through repeated testing in this way we were able to create the hybrid strain shown in Plot 6; a hybrid strain that is easily grown in solid substrate culture, with a potency greater than any other cultivated strain and at least equal in potency to the highest quality wild Cordyceps.
John C. Holliday PhD
Phillip Cleaver BS
Megan Loomis-Powers BS