Culture Jars

Materials

• Original source culture:
  • P. Stipticus 10cc liquid mycelium syringe
  • Dated 2012-11-28
  • Kept refrigerated in household refrigerator from arrival to inoculation
• Vessels: Pint mason jars
• Vents: Tyvek from FedEx envelope. Inset vent approx 1cm circular. Total vent tops jar with Tyvek
• Water: President's Choice natural spring water
• Corn syrup: Crown Golden Corn Syrup, best by 11:35 Oct 25 2015D
• Honey: Doyon Miel Pur, undated, purchased ca. 2013-01
• Molasses: Grandma fancy, preveiously open, BB/MA 2013 OC 05 08:27 SJ
• Fertilizer: Miracle-Gro All Purpose Liquid Houseplant Food 8-7-6 with 0.10% Chelated Iron, at purchased strength
• Mineral: Flora Laboratories "TEMO" orchid mineral supplement: 1/2 kitchen tsp (nominal 2.5 mL, approx. 1.15 to 1.2 g) powder per 100mL solution
• Dowels, type 1: Generic Home Depot "Hardwood Dowell Pin SG", 3/8" x 2"
• Dowels, type 2: 3/8" poplar dowel from Canadian tire, cut into approx 2" lengths using chisel.
• Sealant: GE Silicone I Kitchen and bath
• Vent filter: Tyvek from FedEx large envelope
• Heat source: Bosch kitchen stove running on propane
• Pressure cooker: Kuhn Riken "larger" pressure cooker
• Stock for jars 5, 6, and 7: 1L PC water, 17g corn syrup, 0.35mL TEMO mineral, 0.35mL fertilizer, microwave 1 min to dissolve

Preparation procedure

• Prepare jar lids:
  • Each lid drilled with two holes: approx 1/2 inch for vent and approx 3/16 inch for injection septum.
  • Septa:
    • Cover 3/16 hole from both sides with silicone seal
    • Leave concave top of lid down on wax paper to form flat septum top.
    • Build up material to slight dome on underside of lid (more for jars after 4)
    • Septa approx 1.5cm overall diameter.
  • Vents:
    • Cover 1/2-inch hole with Tyvek from top of lid
    • Tyvek extends at least approx 0.75cm beyond edge of hole on jars 1-4, possibly less on 5-8. Possibly less free "breathing" space on 5-8.
    • Glue Tyvek down around outside edge with silicone seal
    • Add thin layer of additional silicone seal around top of outside edge
• Clean jars (also standard procedure for other glassware):
  • Soak in 50-60C tap water with dishwasher detergent (missed for 5, 6, 7)
  • Rinse thoroughly with tap water
  • Rinse thoroughly with PC spring water
• Prepare media:
  • Place all media, dowels, etc in jars, install lids
  • Quantities measured to "kitchen" standards
  • Jars 1-4
    • PC water measured with 100mL graduated cylinder
    • Syrups measured with AND EK-1200g 1200x0.1g scale
    • Fertilizer and premixed mineral measured with 2mL disposable pipette
  • Jars 5-7
    • Measure 1L PC water in kitchen measure
    • Add 17g syrup with kitchen 22000x1 scale
    • Fertilizer and mineral with 2mL disposable pipette
• Sterlization:
  • Place jars in pressure cooker (4 jars per batch)
  • Use perforated "spacer plate" to separate jar bottoms from cooker bottom
  • Add PC water to fill pressure cooker to jar shoulders
  • Cook at high pressure, plus heat-up time and natural release. Jars 1-4: 30 min. Jars 5-7: 45 min
  • Cool overnight
• Inoculation:
  • Wipe septa with paper towel soaked in 99% isopropyl alcohol
  • Place drop of alcohol on each septum (did I sop it with the towel?)
  • Wipe syringe needle (preinstalled by vendor) with alcohol towel
  • Heat approx 1.5cm at end of syringe needle to red heat in propane flame
  • Inject 1mL of source culture through each septum in turn
  • Cap each septum with approx 3mm blob of extra silicone
  • Replace syringe in original packaging

Detailed log

• 2013-01-12 by 13:00: Filled jars (forgetting dowels), photographed.
• 2013-01-12 13:01: Close lid, turn large front to high
• 2013-01-12 13:02: Move to small right rear burner on high
• 2013-01-12 13:05: ABORT: forgot to add dowels. Add them.
• 2013-01-12 13:07: Back to high heat on rear burner
• 2013-01-12 13:13: Stove out of gas... sigh.
• 2013-01-12 13:17: Back to high heat on rear burner
• 2013-01-12 13:35: Cooker stem up; reduce heat to medium
• 2013-01-12 13:38: Begin preheating flame spreader on left rear burner at medium
• 2013-01-12 13:39: Stem at line 1. Move to left burner with spreader. Audible "boiling" sounds stop.
• 2013-01-12 13:43: Boiling sounds clearly audible and continuous
• 2013-01-12 13:51: Stem at line 2. Turn heat to low.
• 2013-01-12 13:53: Slight venting
• 2013-01-12 14:08: Established running well above line 2, venting continuously.
• 2013-01-12 14:21: Heat off
• 2013-01-12 14:30: Stem at line 2
• 2013-01-12 14:41: Stem at line 1
• 2013-01-12 ~22:30: Open pressure cooker. Temperature about 36C. Dusting of "mineral deposits" observable on jars.
• 2013-01-12 ~22:35: Transfer jars to office cabinet for further cooling. Photograph (poor lighting and blurry).
• 2013-01-13 09:10: Inoculate jars (in kitchen)
• 2013-01-13 ~09:15: Inoculate jars, move back to office cabinet. 22C, 28% to 30% RH.
• 2013-01-13 12:03: Check office cabinet. 22C, 28% RH.
• 2013-01-14 12:11: Considerable vaguely filamentous cruft around dowels at bottoms of all jars. Not sure if more than last night, when there was at least some. 23C, 30% (possibly/probably heated by photo light). Took photo
• 2013-01-17 19:04: 22C, 26% RH. Jar 4 was beginning to cloud last night and now has significant cloudiness near top as well as the "junk" near the dowels. "Rafts" forming on jars 2, 3, and 4, especially 3 and *especially* 4.
• 2013-01-22 ?: Agitated jars by swirling. Had previously picked them up from time to time. Apparently not much more cloudiness.
• 2013-01-26 09:00 appx: Drew liquid from jar 2 for microscopy using 21-ga needle on 3mL syringe. Found little or nothing in two slides using 90X phase illumination. Agitated jar #2 considerably. Possibly more mass around dowels in bottoms of jars; Warren thinks so, anyhow. Microscopy of purchased stock showed numerous slightly ovoid cells somewhat smaller (and considerably rounder) than putative "paramecia" in other stuff. Also some mycelium-like material, but (from memory in evening) more like 30 microns that 5 across.
• 2013-01-26 23:30: Made lids for jars 5, 6, 7, and 8.
• 2013-02-16 11:56: Prepared jars 5, 6, and 7. Placed over high heat
• 2013-02-16 12:22: Pressure cooker stem up
• 2013-02-16 12:23: Start spreader plate preheating at medium heat
• 2013-02-16 12:25: Pressure cooker stem at line 1
• 2013-02-16 12:26: Reduce spreader plate heat to low
• 2013-02-16 12:28: Pressure cooker stem at line 2, move to spreader plate
• 2013-02-16 12:30: Pressure apparently flagging, increase heat
• 2013-02-16 12:31: Move back to naked flame at low
• 2013-02-16 12:44: Pressure cooker stem at about "line 2.5"
• 2013-02-16 13:14: Remove from heat. Approximately "line 3" and has been for a while. Has been venting.
• 2013-02-16 14:06: Status of jars 1-4: have been agitated somewhat every few days. Now considerable cloudiness floating at tops and cloudiness/"goop" around dowels at bottoms of jars: minimal in jar 1, most in jar 2, intermediate (and hard to see) in jar 3, not that great in jar 4. Thermometer says min/max temp have been 19 and 24, min/max humidity 19%/31%. Possibly noticeable evaporation.
• 2013-02-16 17:30: Agitated jar 2 to suspend contents. On microscopy, found many small, isolated, ovoid objects, probably cells, looked like they had nuclei, maybe 2 microns? A few clumps of smaller ovoids: cocci? Some larger, irregularly round, apparently eukaryotic cells. Widely scattered possible clumps of hyphal fragments; rare compared to other material.
• 2013-02-23 ~15:30:
  • Opened jars 2, 3, and 4 under clean-but-not-sterile conditions, extracted dowels and some "scum" from jar 2 (using alcohol-soaked, but wiped, tweezers), reclosed jars.
  • Dowels from at least jars 2 and 3 had distinct "yeast" odor; not sure about/did not check 4.
  • Made smears from dowels and clinging fluid using Q-tips. Microscopy findings much as on previous microscopy of jar 2: dominantly small egg-shaped (slightly pointed) apparently eukaryotic cells, with some other stuff including "junk" and rare potentially hypha-shaped elements. Cells in "scum" from jar 2 were numerous enough to form "shoulder-to-shoulder" areas probably 100 cells wide.
  • Drove all 9 dowels into section of tree limb
    • Limb previously collected from deadfall outside community center around the beginning of February. Species unknown.
    • Approximately 10cm diameter
    • Bark reasonably intact, ends reasonably cleanly cut.
    • Had been frozen, but was inside for about an hour before dowels were driven. Wood very wet.
    • Holes for dowels were 13/32", which resulted in a tight fit.
    • Dowels passed nearly through log
    • Sealed holes with paraffin wax dripped from a candle
    • Three dowels from jar 2 were near end where subsidiary branches had been. Dowels from jar 3 in the middle. Dowels from jar 4 at other end.
• 2013-04-27 14:23:
  • Jar 5: white raft of mycelium
    • Probably 3mm thick with further extensions into water
    • Mycelium threads extending up glass above water line 1 to 3 cm.
    • Visible light in full darkness after ~15 minutes eye adjustment with jar held to eye, probably from threads.
    • On removal, raft was solid mushroom-like tissue, not just mycelium net.
  • Jar 6 (control): appears clear
  • Jar 7: Cloudy. White material on bottom of jar. May have had a raft a few days ago (?).
• 2013-04-27 17:19
  • Cut 3 logs; wood had been sitting in driveway; thawed at least 2 weeks; not wet but not "lumber dry"
  • Plugged one log with jar 7, two with jar 5.
    • Plugs were too long for holes; vaguely tried to cut off most and seal them
    • Tops of may were "fuzzy" from hammering.
    • No attempt at sterile technique.
  • Poured liquid and "raft" from jar 5 over logs in terrarium
  • Covered terrarium loosely with plastic wrap
  • Old log from terrarium beside bench in back yard
  • Liquid from jar 7 dumped on grass at rear of driveway

Random notes

Weight of jar 4 before sterilization was 552.2g, calculated from value shown on scale after removing jar; scale had been tared and 5.1g corn syrup had been added.