An experiment I conducted
Trial 3 (27/09/2010 - 31/11/2010)
The purchasing of materials began on 11/9/2010. Inoculation took place on 27/09/2010, and signs of germination were clearly visible on 01/10/2010. The jars were completely colonized on 11/10/2010, and were spawned onto a pasteurized straw substrate 11/10/2010 – 13/10/2010. Out of the 5 jars that were inoculated, 2 were contaminated with Bacillus bacteria. The remaining 3 jars were spawned into a straw substrate 4 inches deep. A grain master spawn was attempted, however the two jars that were spawned did not show any considerable signs of growth and also became contaminated with Bacillus (Wet Spot). However the straw colonized rapidly, and was fully taken over on 21/10/2010. The casing layer was applied on 23/10/2010, and was deemed colonized on 01/11/2010. Of the four trays placed in the fruiting chamber on 01/11/2010, 2 were seen to be infected with trichoderma. It should be noted that after the application of the casing layer, the environmental conditions weren't monitored closely and visual inspections were performed at a lesser rate. This may have contributed to the trichoderma contamination. All attempts to treat the contaminated casing and substrate were futile (Hydrogen peroxide, excision). Pinheads were seen in tray 3 (the single productive tray for practical purposes) on 06/11/2010, and the first flush was underway the next day. On 31/11/2010, Trial 3 was over in terms of fruitbody production. Overall, only trays 1 & 3 were able to produce fruitbodies. Tray 3 produced approximately 46 grams dry, while tray 1 produced 4 grams.
For future operations, it is highly recommended that all equipment, containers, materials and surfaces to be re-used be disinfected effectively. A re-evaluation of the casing mixture (Peat Moss, Vermiculite, Crushed Oyster Shell, Limestone flour) may be needed to reduce the contamination risk, and it is also worth stating that the quantities of the materials involved should be calculated precisely in any future endeavors. Processes such as pasteurization and casing should be synchronized as not to keep processed materials exposed to potential airborne contaminants for extended periods of time (Example: The casing mixture cooling in an open bucket next to the open window, 22-23/10/2010.) The next operation should focus on producing the maximum amount of colonized grain possible, as grain spawning and substrate colonization must advance concurrently under ideal conditions. As soon as the jars are available, spores must be available and ready for inoculation. Through this methodology, a reliable bank of grain culture can be maintained while simultaneously colonizing straw substrate trays and casing/fruiting grain spawn. The numbers of trays, fruiting chambers and incubators that can be maintained must of course be considered beforehand.
In terms of expected net dry weight, the trial was an abysmal failure; of the 750 – 1400 g dry expected, only close to 50 was obtained. The leading cause for this staggering gap between the yield obtained and that posted as average by scholarly literature was the inability to maintain sterile technique through all procedures required. While the prepared jars were pressure sterilized for longer than the required amount of time, it is certain that the jars and their filter disc structures came in contact with aerial spores of Bacillus at some point following sterilization. During incubation, white mycelial growth soon declined as unmistakeable signs of wet spot contamination became evident in the two jars mentioned. It is possible to construct an inexpensive glove box that can allow the jars to cool and be inoculated. General hygiene, of course, can always be improved. (Vacuum some days before launch of the trial, wipe more surfaces with 10% bleach, do not operate air conditioning, wear medical protective caps, etc.) The 'domes' that were constructed in order to stop water damage to the casing layer were not only completely useless and unnecessary, but they also required much effort to construct. The use of depth rings during the application of the casing layer is also a good idea for the next trial.
