Experiments+detail+log

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=07.2013 - Operation Restart - Details= > - 5 jars of barey grain (200g grain/180g water), soaked 8h, PC sterilized,, used 24h later. among them, one with.0.5cm of finely shredded straw and another with 1cm of vermiculite on top > - spawn : made from a fractured agar plate, barley spawn previously contaminated with trychoderma, but was cleaned (contaminated grain removed), kept in non-sterilized condition and recovered, used at almost full double colonization after 5/6 weeks > - inoculation of new grain with 10% home made spawn in glove box, use of peroxyde mist to mitigate contaminants spores > - glove box sterilization : presoaking in alcohol spray 75°, wait 20min with aquarium pump pushing air inside through tivek, operation with alcohol 75° sterilized tools, misted peroxyde ( 30% of commercial peroxyde (of unknown concentration)) when spawn was open, and before each jar > - incident during operation (after 2 jars), gloves teared along (2 holes of 1cm each), fastely repaired with tape > - jars set to rest at 30° > - 2 mini jars used as control, one of them was opened and a alcohol sterilized paper placed inside to test for glovebox sterilization (presence of airborne spores). Another was jept closed to test for sterilization process
 * 1) **Spawn Making Hyspizygus (potential Pleurotus pulmunarius) G2G - 24.07.2013**
 * Substrates**
 * Procedure**
 * Result :** after 5 days, colonized at 40%, no contamination. Straw and especially vermiculite covered was late at start, but seem to catch on others.
 * Discussion :** after 5 days, the expected contaminants from spawn didn't develop yet, it has probably been outcompeted, harmed by peroxyde, or alrerady digested in the spawn by mushroom mycelium

> - 5 jars of barey grain (300g wet), prepared with the RR method, stored in the freezer for several days and 4/5 of them resterilized without aluminium cover and used 24h later. > - Hypsizygus ulmarius entirely colonized MYPA Agar (No 11) petri (9cm) inoculated with 10 drops of spores the 15.8.2013 > - glove box sterilization : presoaking in alcohol 75° spray, wait 20min with 2 aquarium pump pushing air inside through tivek, operation with alcohol 75° sterilized tools > - jars cover untighting > - petri opening and agar division in five pieces > - agar pieces where placed in each of the five jars, agar side touching the grain, without shaking > - no control used
 * 2. Master Grain Making 28.08.2013 - ** Inoculation of Hypsizygus ulmarius MYPA Agar on 5 jars of grain
 * Substrates**
 * Procedure**
 * Result :** waiting result
 * Discussion :**

= = **Operation Sunrise - Details - 03-06/2012** (last update 18/03/2012)

I - Spores
> ===1. Hypsizygus ulmarius=== > ===2. Pleurotus pulmonarius=== > ===3. Pleurotus djamor===
 * Substrates :** 3 Autoclaved Agar : PDYPA in jar, MYPA in jar, Antibiotic (unkown base) in petri, Autoclaved Rye Grain in jar.
 * Procedure :** Inoculation in glovebox, from Syringe (from MRCA Shop), with cloud of pregerminated spores. Incubated at 18-22C. From 0.25 to 1 ml.
 * Result :** PDYPA : received the cloud developed first, white few mm dot in 2 days, after 1 week, half of the agar and additional germination of spores. Antibiotic : Many germination after 5 days, MYPA : many spores germination after 1 week. Grain : germination after 1 week.
 * Discussion :** Successful, with more than 4 germinated spores per cm2. The spore dose should be reduced to allow sectoring. Higher temperature is desirable.
 * Substrates :** 4 Autoclaved Agar : PDYPA in jar, MYPA in jar, 2 Antibiotic (unkown base) in petri, Autoclaved Rye Grain in jar.
 * Procedure :** Inoculation in glovebox, from Syringe (from MRCA Shop), with cloud of pregerminated spores. Incubated at 18-22C. From 0.25 to 1 ml, with 2 drops and 0.5ml for antibiotic petris.
 * Result :** PDYPA, Antibiotic, MYPA : many spores germination after 1 week. 2 drop Antibiotic : enough germination, 0.5ml Antibiotic : too many germinations. Grain : received the cloud, germination after 1 week.
 * Discussion :** Successful, with more than 2 germinated spores per cm2. The spore dose should be reduced to allow sectoring. Higher temperature is desirable. Surprisingly, the 1ml petri was slower to germinated and stopped growth after 2-3mm, and is, as such, unusable for sectoring or transfer to grain. This can be due to the inhibitory effect of germinated spores on each other[reference needed].
 * Substrates :** 3 Autoclaved Agar : PDYPA in jar, MYPA in jar, Antibiotic (unkown base) in petri.
 * Procedure :** Glovebox operations. From Self harvest very fat spore print with probable contaminations, hydrated in 6ml of sterilized water for 14h into a pinkish solution. 3 ml for antibiotic agar and 1.5ml for others.
 * Result :** No growth in 2 days
 * Discussion :** waiting later result

II - Cropping
> ===1. Pleurotus eryngii===
 * Substrates :** Rye grain jars, Vermiculite + wheat bran jar, Coffee + bran (10%), Woodchips + bran (33%) and Woodchips + straw + bran (35%) in jars and 5l oven bag. All autoclaved for 1h for Woodchips, and 20 min for grain, with 75% water (3x dry substrate).
 * Procedure :** Glovebox operations. From grain spawn from MRCA Shop, 10-20% of substrate weight.
 * Result :** Fuzzy growth in 2-3 days. 2 Grain jar has been accidently excessively watered, and partially clamped.
 * Discussion :** Water seems insufficient for vermiculite and woodchips mixes waiting later result.

=09.2011>06.2012 - Operation Spore Revival - Details -= (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 : no success
**Substrates :** straw, sawdust and cardboard, wheat grain, agar **Procedure :** Huge amount of spores, probably contaminated. **Result :** straw, sawdust, cardboard : no growth. wheat, agar : contamination **Discussion :** Spores too dry. Ziplock bags are not air tight. Bulk substrates (straw, sawdust, cardboard) insufficiently nutritious for germination. Substrate and procedure insufficiently sterile.

2. Germination on PDA : very little success
**Hypothesis :** Autoclaving and agar are necessary for spore germination **Substrates :** 3 autoclaved PDA agar **Procedure :** Spores pre-hydrated 12h, from print contaminated. Germinated spores are transferred. **Result :** 1 agar with germination of 5mm with green mold, collapsed after transferr to inadequate Agar (too humid, didn't solidify). 1 agar with green mold alone and 1 agar with no trace of any growth, probably contaminated by bacteria. Growth, of mycelium and fungi, stops early. **Discussion :** Autoclaving and agar does not prevent from contaminants on spore print itself. Too many bacteria and fungi competition

3. Germination on Antibiotics PDYPA : very little success
**Hypothesis :** Antibiotic necessary for contaminated spores germination **Substrates :** 3 Autoclaved Agar : PDYPA in jar, MYPA in jar, Antibiotic (unkown base) in petri. **Procedure :** Glovebox operations. From Self harvest very fat spore print with probable contaminations, hydrated in 6ml of sterilized water for 14h into a pinkish solution. 3 ml for antibiotic agar and 1.5ml for others. **Result :** In antibiotic agar, green mold after 1 week, beginning of germinations stopped after 5 mm at best, similarly to step 2. After 2 weeks agar changed changed color, orange spots on the surface. Non-antibiotics agar show no growth and no mold, 1 show orange coloration, both probably contaminated **Discussion :** Antibiotic necessary but not sufficient. Spores too old and dry. Quantity of spores is too high (see Sunrise Operation). Too much bacterial contamination or too few antibiotics that worked only 10 days. Too many mold spores with ph too acidic, fostering mold growth.

3. Germination on Antibiotics PDYPA enriched and adjusted
**Hypothesis :** Fewer spores with active carbon and basic ph help germination. Cinnamon antibacterial and anti-mold effect. **Substrates :** 7 Autoclaved Agar : 1 Antibiotic unkown base, 6 PDYPA with 1/Active Carbon, 2/Salt, 3/Chalk, 4/Baking powder, 5/Gypsum, 6/Cinnamon and 7/Mix of all previous. **Procedure :**Glovebox operations. Fat spore print with probable contaminations, hydrated into 10ml blurry solution for 14h, in sterilized water with active carbon, 2g/l salt and 50mg/l cefixime antibiotics. **Result :** waiting result **Discussion :** waiting result

=Operation Dawn - Details - 12/2011-03/2012= (last update 18/03/2012)

I - Clones
> ===1. Pleurotus eryngii clones on agar and grain=== > ===2. Pleurotus eryngii clones on straw and cardboard=== > ===3. Pleurotus ostreatus (possible Hyspizygus) clone=== > ===4. Agaricus bisporus clone=== > ===5. Shiitake clones===
 * Substrates :** autoclaved agar, non autoclaved agar, autoclaved rye grain, autoclaved millet grain (3 weeks control)
 * Procedure :** Caps from supermarket. Glovebox operation. Agar and grain in jars. Successful agar was transferred to grain. Rye grain, but not millet, was shaked many times, loosened at inoculation, filled only half the jar.
 * Result :** Non autoclaved agar : very strong growth in 3 days, but contamination. Autoclaved agar : thin growth and no contamination, full agar colonisation and micro pins after 1-2 weeks. Grain : direct clone has best growth, transfer from agar autoclaved or not was successful also, 2-3 weeks for full colonisation. Millet (transferred fro non autoclaved agar) show stop of colonisation at mid jar.
 * Discussion :** Millet too dense. Rye perfect density, quantity too low.
 * Substrates :** pasteurized cardboard, autoclaved cardboard, straw
 * Procedure :** Caps from supermarket.
 * Result :** Pasteurized cardboard, fast growth, full colonisation in 5 weeks, Pinning in 7 weeks, partially infested by fungus gnat larvae.
 * Discussion :** Unsufficiently protected from fly. Autoclave loosen cardboard structure, but necessary for later contamination.
 * Substrates :** Rye grain autoclaved
 * Procedure :** Caps from supermarket, tissue transferred and jar incubated at 16-18C. Tape was replaced by micropore at week 3. Glovebox operations.
 * Result :** Slow development, colonization stopped at top half after 6 weeks. Green mold appeared and disappeared. Reddish liquid suggest mycelium fighting contamination.
 * Discussion :** Probably contaminated, grain was or too wet or too compact.
 * Substrates :** Rye grain autoclaved
 * Procedure :** Caps from supermarket, tissue transferred and jar incubated at 16-18C. Tape was replaced by micropore at week 3. Glovebox operation.
 * Result :** Very slow development. Growth from decaying mushroom. Almost full colonisation at week 6. Very rizhomatic and thin mycelium.
 * Discussion :** Grain probably too compact. Few inoculation points.
 * Substrates :** Agar
 * Procedure :** Caps from self harvest and old spawn from 2 weeks bulk culture, surely contaminated. Transferred to agar 1 time. Glovebox operations.
 * Result :** Only spawn still generate growth of few mm. Green dots on agar.
 * Discussion :** Too many contaminant, spawn too old, can be transferred again.

II - Cropping
> ===1. Macrolepiota procera===
 * Substrates :** Straw
 * Procedure :** Pasteurized straw, incubation in large boxes, with coffee filter lid. Grain spawn 6 month old in the fridge.
 * Result :** Good growth, 1-2 month for full colonisation. No contamination. At full stage, infested with fungus fly larvae, treated with nematodes and hypoaspis, that partially contaminated the surface.
 * Discussion :** Better separate from fungus fly larvae (from other bags). Ready for outdoor bed inoculation.

III - Spore
> ===1. Shitake spores collection===
 * Substrates :** Agar, Aluminium foil.
 * Procedure :** Spore collection from self harvest. Directly on Agar, Aluminium foil under living mushroom (insitu collection).
 * Result :** No spores
 * Discussion :** Fungus fly larvae, sporeless caps, too old culture.

=Operation Paddle - Details - 08-12/2011= (last update 18/03/2012)

I - Crop (see Photos)
> ===1. **Pleurotus djamor** : Major success, with some failure at the end=== > ===2. **Lentinula edodes** : Success, with some failure at the end=== > ===3. **Macrolepiota procera** : Failure with some success at the end===
 * General :** Mycelium growing in the bags in 3 phases : 1/ white cotton growth, 2/ myclium is saturating the bags, ryzomatic like growth, kind of cheese, 3/ mycelium seems regressing, the substrate get more visible (browning for Shiitake). 4/mycelium converge to form primordia into bags hole or more aered areas. Each mycelium change the color of the substrate, in a particular way. Pleurotus djamor djamorizes the straw and woodsaw into an orange color, while Lentinula edodes gives more a brownish color.
 * Substrates :** Straw, Hay, Straw + Woodchips. Straw pellets, Straw Pellets + Paper + Cardboard, Woodsaw + cardboard + paper.
 * Procedure :** Pasteurized substrate in 8l bags, with fresh grain spawn.
 * Result :** Successful - Straw + Woodchips (8L & 30L bags): quite fast (10 -15 days) and compactedness seems perfect, Straw: Fast to produce (10-15 days). Less successful - Hay : Similar to Straw, a bit slower and more contaminated, Straw Pellets + Paper + Cardboard : Slow (25 days) and quite compact, Straw pellets : Very slow (30 days) and very compact, few aborts, Woodsaw + cardboard + paper : no primordia after 25 days and colonisation regressing. Paper + Cardboard + Hay + Straw + Plant waste + Straw Pellets : Similar to straw, slower and more contaminated, very compact. All bags has been invaded by fungus fly, especially later and larger ones.
 * Discussion :** structure of the substrate is the key, and dont excessive water. Fruiting chamber must be better isolated.
 * Substrates :** Straw, Woodchips, Woodchips + Straw, Woodchips + Cardboard, Woodchips + Straw pellets.
 * Procedure :** Pasteurized substrate in 8l bags, with fresh grain spawn. Outdoor buried one time.
 * Result :** Woodchips + Straw pellets, and Woodchips + Cardboard : Fast growth, Full colonization and thick colonisation after 30 days. Woodchips : Half colonization after 15 days, slower growth. Woodsaw + Straw : Half colonization after 15 days, growth more in compact areas. Straw : Fast to produce, primordia after 30 days, but more easily contaminated. Bags half colonized, other half contaminated. 50% of bags reached only half colonisation. After 3 month, fungus fly infested the culture substrate. After 5 months, humidity loss dried totally the substrate, humidification only triggered wold growth. Outdoor buring attempt resulted in 2 nice mushrooms.
 * Discussion :** Wood chips with finer material is best as substrate. Cultures must be isolated and hydrated, on the long run. Dry bags can be buried in new substrate.
 * Substrates :** Straw, Hay, Straw pellets, Straw + Soil.
 * Procedure :** Pasteurized substrate in 8l and 2l bags, with fresh grain spawn.
 * Result :** Woodchips: Growth, fully colonised in 3 months. Straw + Soil : Growth, fully colonised in 2 months, with black mold on soil dense part. Straw : slow growth, contamination, contact colonized by pleurotus djamor untill fruits/aborts, easily contaminated by Fungus Gnats. Hay : slow growth, contaminated, but with many contaminants (green mold, cobweb mold, unkown). Straw Pellets : no growth, contaminated, too compact. All bags were easily contaminated and attracted fungus gnat even in early stages.
 * Discussion :** Better isolation of fruiting bags. Better sterilization. Slow mushroom need better sterlization

II - Clone (photos)
> ===1. Agaricus bisporus : failed=== > ===2. Pleurotus ostreatus (potentially Hypsizygus ulmarius) : success at the start and than failed=== > ===2. Pleurotus eryngii===
 * Substrates :** Agar pasteurized
 * Procedure :** Organic from buttons and mature caps from supermarket. Spores transferred on agar plastic bags and jar.
 * Result :** No growth for button and plastic bags, very slow and thin growth for portobello in the jar, agar covered in 3 months.
 * Discussion** **:** Ziplock backs are inappropriate and not air tight. Agar quantity too small in jar.
 * Substrates :** Agar cardboard then carboard then straw, woodchips
 * Procedure :** In ziplock plastic bags. Step 1: Cloning on PDA fresh cap from bulk supermarket. Step 2: Transfer mycelium to carboard and agar. Step 3: transfer to straw and woodchips in 2l bags.
 * Result :** Step 1, slow growth but contamination (after 4 days). Step 2, growth with fewer contamination for agar, and no contamination for cardboard. Step 3, no sucess, slow growth stops and regress, fungus fly contamination.
 * Discussion** **:** Too slow and contaminated substrate
 * Substrates :** Low sugar PDA, Cardboard, (Toilet) Paper, Nothing (cf. CloneufTek), then to Woodchips and Straw mix.
 * Procedure :** Fresh caps from supermarket. Step1: Transfer to substrates in ziplock bags. Step 2: Transfer from cardboard and nothing to Cardboard, Woodchips and Straw mix in 2l bags.
 * Result :** Step 1: After 2 days, PDA, growth in aerial zones without contamination. Cardboard, Paper, Nothing, higher growth. Step 2: after 1 week, Straw only show growth, show dense mycelium after 3 months (at outdoor temperature) but fungus fly larvae.
 * Discussion** **:** Substrate not rich enough and insufficently sterilized.

III - Spores
> ===1. Agaricus bisporus Spores inoculation : 0% success=== > ===2. Pleurotus djamor Spores inoculation : 0% success===
 * Substrates :** PDA, Wheat grain
 * Procedure :** Agaricus bisporus Buttons under plastic wrap bought at supermarket the delivery day (checked the store every morning). Due to the position of the caps, a large sporeprint was on the plastic under the caps. No other spores got from caps after 48h. Spores deposited dry on the substrate.
 * Result :** Bacteria on agar, Cobweb on Wheat.
 * Discussion :** Plastic ziplock is not air tight, boiling and even tyndallization is unsufficient. Spores too dry. Insufficient air exchange flow.
 * Substrates :** straw, sawdust and cardboard, wheat grain, agar
 * Procedure :** Huge amount of spores, probably contaminated.
 * Result :** straw, sawdust, cardboard : no growth. wheat, agar : contamination
 * Discussion :** Spores too dry. Ziplock bags are not air tight. Bulk substrates (straw, sawdust, cardboard) insufficiently nutritious. Substrate and procedure insufficiently sterile.