Experiments+Log

See also Experiments details  See also Experiments Strategy toc =Operation Everywhere=
 * ** A. Spore Germination **
 * ** 05.01.14 : ** P.O Sectoring from P1 to 2P2, transfert from a contaminated P1 to P2, all antibiotic agar
 * ** 15.12.13 ** :Spore germination from bought wild mushroom, probably P. ostreatus, on 2 antibiotic agars. The print was on a mushroom cap underneath. Agar petris poured after sterilization, before fanning glovebox.
 * ** 16.11.13 ** : Test spores from our own (late) mushroom of P.pulmonarius on antibiotic agar. The spore print is very thin, almost unnoticeable by eye. The agar was sterilized tilted, thus is not horizontal. By 27.11.13, many germination are visible, but it is not yet sure it is not contaminated.
 * ** 05.11.13 ** : Test viability of new spore syringes, after frost, of P. pulmonarius, A.aegerita, S. rugoso-annulata, A. bisporus and H.ulmarius, 3 drops on 1 agar each. By 10.11.13, Strophoria has many germinations and Pioppino started to germinate. By 13.11.13, Hypsizygus ulmarius started to germinate, despite water in its plate. By 15.11.13, P. pulmonarius started to germinate, despite water in its plate.
 * **30.10.13** : Pleurotus pulmonarius new spore syringe germination on 3 P0 agar (2 new & 1 resterilized old used for failed germination). By 07.11.13, the 2 new agar show new germinations.
 * **30.10.13** : Agaricus blazei-murrill spore syringe germination on 2 P0 agar (1 new & 1 resterilized old used for failed germination) . By 13.11.13, no sign of germination.


 * **B. Agar to Agar & Cloning**
 * ** 05.01.14 : ** L.E commercial dehydrated spawn to 2 agars
 * ** 16.12.13 : ** Cloning of bought wild mushroom, probably P. ostreatus. on 2 antibiotic agars. Agar petris poured after fanning glovebox.
 * **15.12.13** : Cloning of bought wild mushroom, probably P. ostreatus. on 6 antibiotic agars. Agar petris poured after sterilization, before fanning glovebox.
 * ** 20.11.13 ** : A. aegerita, P.pulmonarius, S.rugoso-annulata, A. bisporus & H.ulmarius transfer from P0 (A.05.11.13) & P.P. from P0 (A.30.10.13), to new agars: 2 P.P. (A05.11.13), 2 P.P. (A30.10.13), 3 A.E., 1 S.R, 1 H.U. & 1 A.B.
 * **20.11.13** : P. citrinopileatus transfer from P+0 (donia clone) to 1 antibiotic agar & from P+1 to normal agar.
 * **20.11.13** : G. lucidum transfer from P+1 (B.30.10.13) to antibiotic agar.
 * ** 08.11.13 ** : Test again viability of stored Hypsizygus P0 agar, before slant storage, by transfer to 1 new agar. By 13.11.13, show growth.
 * ** 08.11.13 ** : Saving of P.C. by transfer from all agars (1 mother culture, 2 P+1, 1 possibly misnamed P+1 & 1 mold contaminated P+1) to 5 new agars. By 13.11.13, show some growth, but seems weak.
 * ** 05.11.13 ** : Test viability of stored Hypsizygus P0 agar, before slant storage, by transfer to 1 agar. By 13.11.13, show growth despite, very few agar and water.
 * ** 30.10.13 ** : P. citrinopileatus, P. eryngii, P.ostreatus & Ganoderma lucidum donia original clones transfer to new agar. By 07.11.13, P.E. & G.L. fully colonized, but P.C. slowly growing & P.P. no growth at all. P.C. & P.P. Possibly adversely affected by storage, especially accidental freeze.
 * ** 30.10.13 ** : P. eryngii clone (transferred from donia clone) transfer from P+1 antibiotic to 2 new P+2 non antibiotic agar. By 07.11.13, agar fully colonized. 1 is used for transfer to slant.


 * ** C. Agar to Slant **
 * **20.11.13 :** H. ulmarius transfer from P0 to 2 tube & PC from P1 to 1 tube. Tubes agar was a bit too liquid, again.
 * **08.11.13** : P.E & G.L. P+2 agar, from B.30.10.13, transferred to 2 slant. By 13.11.13, slant fully colonized. At 20.11.13, the too much water was emptied.
 * **05.11.13** : P.eryngii P+2 agar, from B.30.10.13, transferred to a bit too liquid slant. By 13.11.13, growed on left agar, was turned upside down to avoid water.


 * ** D. Spawn Making **
 * **07.12.13 :** P.P 2 jars master grain from 1 agar, G2G: P.E 8 jars G1 from 1 jar master grain, H.U 2 jars G2 to 10 jars and 3 1kg bags G3
 * **23.11.13** : G2G transfer of H.U. : one jar G1 to 3 jars and one bag 800gr G2 and from one jar G2 to 3 jars and one bag 800gr G3,
 * **23.11.13** : PC et PP master grain from agar P+1 & P0, respectively.
 * **17.11.13** : G2G transfer of one H.U. G1 jar, from D.29.10.13, to 5 new jars and a 800gr bag of freshly prepared barley (RR method).
 * **17.11.13** : Grain master making from P.eryngii agar (probably from G3 of operation Restart) to 4 jars of freshly prepared barley (RR method)
 * **16.11.13** : G2G transfer of one H.U. G1 jar, from D.29.10.13, to 5 new jars and 1 1.5kg bag of freshly prepared barley (RR method). The bag bottom accidently broke outside glovebox during shaking and was fastly taped. By 18.11.13, all jars & bag start to grow. By 27.11.13, all jars are colonized, & the bag is half colonized only and seems to stop (is the bottom contaminated ?).
 * **09.11.13** : G2G transfer of one H.U. G1 jar, from D.29.10.13, to 5 new jars (kept frozen & resterilized, 1 of them was burned pearled barley) and 1 bag (had mold mycelium traces, kept frozen and resterilized). By 13.11.13, 4 of 5 show good growth the other show only recovering, the bag has a start of growth.
 * **29.10.13** : G2G transfer of one Hypsizygus ulmarius home made master grain (from Operation Restart E.) to new 5 jars (300gr wet each) and 1 bag (1kg wet) of barley prepared according to RR method. By 07.11.13, all jars and bag are fully colonized, ready for use or storage. By 13.11.13, the bag has 2 spots of 2 cm, that don't want to colonize, potentially bacterial contamination. By 15.11.13, in the bag the spots were not colonized and took a blackish slimy appearance, seems to confirm bacterial contaminant.


 * **E. Fructification**
 * **27.11.13** : Hypsizygus ulmarius inoculated from G2 on straw bags, prepared with RR method. 6 bags of average 9 kg (wet weight) were inoculated with spawn at rate average of 25% of dry straw weight.
 * **21.11.13 :** Hypsizygus ulmarius inoculated from G1 & G2 on straw bags, prepared with RR method. 10 bags of 1 kg (wet weight) & 1 bag of 10kg (wet weight) were inoculated with spawn at average rate of 5% of wet straw weight.
 * **15.11.13** : 8 bags of straw, prepared according to RR method, where inoculated with H. ulmarius spawn (the bit contaminated bag from spawn making operation D.29.10.13), with average 100gr of spawn for 1000-1200.gr of wet straw. 2 bags where inoculated at double rate with 200gr for 1 kg and 100 gr for 500gr wet. The bags were placed in the newly prepared cellar.

=Operation Restart - 05.2013 to 09.2013=
 * [[image:IMG_5641.JPG height="188" caption="D. Spore germination. One petri after one week" link="@file:mushroomway/IMG_5641.JPG"]] || [[image:mushroomway/IMG_5667.JPG height="188" caption="F. Sectoring 14 days later. Original agar after the 5 transfers" link="@file:mushroomway/IMG_5667.JPG"]] || [[image:IMG_5662.JPG height="188" caption="E. Master grain making. The 5 jars with their piece of agar, after operation" link="@file:mushroomway/IMG_5662.JPG"]] ||  ||
 * **G. Agar to agar**
 * 1) ** 02.09.13 ** : Transfer of Donia clones (eryngii, citrinopileatus, pulmonarius & Ganoderma lucidium), to 20 small petri made by Donia & 4 large petrimade by us. After 24h, all was contaminated by bacteria (ours mostly at the border).
 * 2) ** 04.09.13 ** : New transfer from Donia clones (eryngii & citrinopileatus) to 4 antibiotic petri. After 3 days, eryngii recovered and started to grow, but not citrinopileatus. H2O2 was used during operation, which may have stunned growth.
 * 3) ** 05.09.13 ** : Transfer of G1 clones (eryngii & citrinopileatus) away from contamination. After 2 days, eryngii recovered and started growing on the new media, but not citrinopileatus. The petri used were particularly humid (from the pressure cooker) with some water inside, which may have slowed growth.
 * **F. Sectoring 29.08.2013** - Transfer of Hypsizygus ulmarius MYPA agar sectors (from op. D) to 5 new MYPA agar to isolate strains
 * ** E. Master Grain Making 28.08.13 ** - Inoculation of Hypsizygus ulmarius from MYPA Agar (from op. D) on 5 jars of (RR style) grain. After 10 days, 30% colonized, the grain appear a bit dry, probably due to 30 - 35° ambient temperature.
 * **D. Spore Germination 15.08.13** - Inoculation on MYPA Agar of Pleurotus pulmonarius & Hypsizygus ulmarius syringe (5 & 5 dishes). Several drops conditions, 2, 5 & 10 drops. Kept at 33 - 35°C. After 6 days, germination on 2 plates over 5, but no germination yet for Pleurotus.
 * **C. Spawn Making G2G - 07.13 to 08.13**
 * **05.08.2013** - 4 pearled & 1 burned whole barley jars with homemade Pleurotus pulmonarius spawn (that recovered from contamination & kept in unsterile condition), peroxyde not used during operation. Incubation at more than 33°, few days at 38°. After 2 weeks, 4 over 5 jars contaminated (black mold). 1 jar (burned barley) completed by 11 august. Incubation probably too warm, starting spawn contaminated.
 * **24.07.2013** - whole barley inoculated with homemade Pleurotus pulmonarius spawn (that recovered from contamination & kept in unsterile condition), peroxide used during operation. Incubation at more than 33°. After 5 days 40% colonization, no contaminant. After 2 weeks, 2 over 5 jars contaminated (green mold, trichoderma). 3 jars completed by 11 august. Incubation too warm, starting spawn contaminated.
 * ** B. Indoor Fructification **
 * **06, 11 & 14.08.13** - Homemade Pleurotus pulmonarius spawn (from op. C) on pasteurized straw, in 2, 10, 4 & 4 plastic bottles. 2 spawning level, 10-15% & 80-100% of dry weight (3-5 & 15-20% wet straw weight). Those started the 6.08.2013 (10 - 15% spawn level), started to pin out the 24th and released the spores the 30. Those started the 15 (80%-100%) started to pin the 30.
 * **05.2013** - Commercial Pleurotus eryngii spawn on sterilized coffee ground, in hermetic bags. After 3 months 20% colonization. Substrate too dry, too compact & too little fresh air exchange
 * **05.2013** - Homemade Pleurotus pulmonarius spawn on sterilized coffee ground & straw, in hermetic bags. After 3 months, 80% straw & 50% coffee colonized. Substrate too dry (& too compact for coffee), insufficient fresh air exchange
 * [[image:IMG_5602.JPG height="188" caption="B. Mycelium running, after few days" link="@file:mushroomway/IMG_5602.JPG"]] || [[image:IMG_5645.JPG height="188" caption="B. Primordia formation, around the holes" link="@file:mushroomway/IMG_5645.JPG"]] || [[image:https://fbcdn-sphotos-c-a.akamaihd.net/hphotos-ak-prn1/1236577_10151850945846031_1818517361_n.jpg height="188" caption="B. Pinning out, j+ 21"]] || [[image:https://fbcdn-sphotos-h-a.akamaihd.net/hphotos-ak-prn2/1235420_10151860765136031_2044197992_n.jpg height="188" caption="B. Ready to harvest, j+25" link="@https://fbcdn-sphotos-h-a.akamaihd.net/hphotos-ak-prn2/1235420_10151860765136031_2044197992_n.jpg"]] ||
 * **A. Outdoor Fructification - 13.08.13** - two patches of pasteurized straw, sandwiched in cardboard, inoculated with contaminated homemade Pleurotus pulmonarius or Hypsizygus ulmarius. After one week 20-30% colonized. By 27.10.13, all straw was dry no sign of mycelium, after one and half month with no irrigation and very few rain.

=Operation Spore Revival - 09.2011 to 06.2012= (last update 28/03/2012) Germinate and purify contaminated spores of home grown Pleurotus djamor. This operation include actions from Sunrise, Dawn and Paddle > **1. Germination on Various Substrates** : Spores too dry. Ziplock bags are not air tight. Bulk substrates inappropriate. Substrate and procedure insufficiently sterile. > **2. Germination on PDA :** very little success. Too many bacteria and fungi competition, must use antibiotics > **3. Germination on Antibiotics PDYPA :** very little success. Antibiotic necessary but unsufficient. Spores too old, quantity too high (see Sunrise Operation), with ph not enough basic. > **3. Germination on Antibiotics PDYPA :** Early addition of antibiotics, addition of active carbon and higher ph. Waiting result

=Operation Sunrise= (02-06/2012) > **Spore** >> **1. Hypsizygus ulmarius :** 0.25ml of spore syringe is much. germination on all agars (PDYPA, MYPA, Antibiotic), after 5 days, germination on grain after 1 week. >> **2. Pleurotus pulmonarius :** 2 drops of spore syringe is enough, 1ml is way too much. germination on all agars (PDYPA, MYPA, Antibiotic), germination on grain, after 1 week >> **3. Pleurotus djamor :** little germination after 2 weeks. Too many spores and contaminants > > **Crop: Pleurotus eryngii** : fuzzy balls, substrate dry after 1 week. waiting more result

=Operation Dawn= (12/2011-03/2012) > **Clone** >> **1. Pleurotus eryngii clones on agar and grain** : Well colonised grain, Millet density high. Rye Quantity low. Waiting fruiting phase. >> **2. Pleurotus eryngii clones on straw and cardboard** : Some success, waiting full colonisation. Protect from flies and contaminants. >> **3. Pleurotus ostreatus (possible Hyspizygus) clone** : Grain too compact, mature but half colonized, probably contaminated >> **4. Agaricus bisporus clone** : Successful, grain too compact. Waiting casing. >> **5. Shiitake clones** : failed. Transfer still possible but risky. > > **Crop: Macrolepiota procera** : fully colonized, waiting for outdoor beds, fungus fly larvae probable. > > **Spore: Shitake spores collection** : culture too old

=Operation Paddle= (08-12/2011) > ==Crop (photos)== > **General Observations :**Excessively watered, that influenced smell & contamination. Bags unsuffiently protected from contaminants and flies (Vermicompost bin at only few meters distance). Fungus fly invaded the bags, beware of their larvae
 * 1) **Pleurotus djamor with pasteurized substrates - Good** : Straw, Straw + Woodchips. **Bad** : Hay, Straw pellets, Straw Pellets + Paper + Cardboard, Paper + Cardboard + Hay + Straw + Plant waste + Straw Pellets, Woodsaw + cardboard + paper
 * 2) **Lentinula edodes (Shiitake) -** **Good**: Woodsaw + Straw pellets, Woodsaw + Cardboard, Woodsaw + Straw, Woodsaw. **Bad** : Straw.
 * 3) **Macrolepiota procera - Good** : Straw, Woodchips. **Bad** : Hay, Straw Pellets, Straw + Soil.

> ==Clone (photos)== > **General Observations :**Use more selective substrate less prone to contamination. Use sterile procedure on Agar. Use fresher possible tissue from a long shelf life mushroom (e.g. P.eryngii, L.edodes). When possible, avoid supermarket diluted genetics fruits.
 * 1) **Pleurotus eryngii**. Good : agar, grain, cardboard. Bad : straw, unsterilized
 * 2) **Lentinula edodes.** Failed. Contaminated and unvigourous fruit.
 * 3) **Agaricus bisporus.** Good : agar.
 * 4) **Pleurotus ostreatus.** Good : straw, cardboard

> ==Spore== > **General Observations :**Pre-hydrate spores. Use agar, preferably with antibiotics. Success as much as error are needed for learning. = =
 * 1) **Contaminated spores of Pleurotus eryngii**. Agar : germination+contamination, dead on transfer. Grain, cardboard, sawdust : contamination only.
 * 2) **Agaricus bisporus.** Failed. Not enough spores, inappropriate technique.

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