NLB inoculum prep guide

Tyr Wiesner-Hanks

July 8, 2015

General timeline

March

  • Find/collect/purchase all the materials you will need:
  • 300 spawn bags from Fungi Perfecti
  • Heat sealer
  • Small autoclave bags, at least 200
  • 20-25 gallon jugs
  • Tween 20
  • HandySteps and HandyStep tips
  • All reagents for LCA medium
  • Ensure HandySteps and heat sealer are working properly

April

  • Make 10-15 plates of NY001 on LCA from frozen stocks

May

  • From 10 started plates, make ~50 plates for inoculating sorghum
  • Start 10 more plates from frozen stocks to make plates for liquid inoculum

June

  • From 50 started plates, inoculate the sorghum bags and shake/break up clumps daily
  • From 10 started plates, make ~500 new plates for liquid inoculum

July

  • Inoculate

Liquid inoculum

Basic outline

Transfer glycerol stocks to 10-20 plates and let grow for 3-4 weeks. Use those plates to make more plates.

Pouring the plates

S. turcica is best cultured on lactose casein hydrolysate agar (LCA). The recipe for 1L is as follows:

ddH2O (ml) / 1000
MgSO4 (MgSO4·7H2O) / 0.5 g (1.022 g)
KH2PO4 / 1 g
Casein hydrolysate (acid) / 1.5 g
Casein hydrolysate (enzymatic) / 1.5 g
Agar / 15 g
Lactose / 37.5 g
Microelements / 2 mL
1M NaOH / 2.75mL

Microelements are made with the following recipe:

Fe(NO3)3·9H2O723.5 mg

ZnSO4·7H2O439.8 mg

MnSO4·4H2O203.0 mg

  1. Mix all the ingredients with 800 ml of water, add H2SO4 until the solution is clear, then add water to a final volume of 1 L.
  2. Ajust pH to 6.0 with H2SO4.

Pouring plates

  1. Mix all of the above ingredients besides the agar
  2. Resultant solution should have a pH of around 4. Adjust the pH up to 6.0 with 1.0M NaOH (takes around 2.75mL to adjust 1L, 2.2mL to adjust 800mL)
  3. When pH is adjusted, add agar and autoclave for 40min liquid setting
  4. When liquid is cool enough to hold indefinitely (about 30-40min later), pour 15-20mL into each plate. Keep other bottles warm at 65C in a water bath when not using.

Liquid inoculum

Basic outline

Fill spawn bags (or milk jugs) with sorghum grains and water. Soak, sterilize, and inoculate with scrapings from a plate.

Prepping bags

  1. Fill spawn bags with 1000mL sorghum and 600mL diH2O. We have two containers cut down to 1000mL and 600mL for this purpose in the glassware cabinet
  2. Make three seals to make an S shape in the top of the bag. Space them out.
  3. Soak overnight
  4. Run bags in the autoclave, 70 minutes liquid cycle. Four bags will fit to a large autoclave bin, one to a small bin. The tops of the bags should all be pointed in the same direction. It works better to just autoclave at night and do 40. One run at 4pm, another around 6pm, another around 8pm, another around 10pm.
  5. Let the bags cool fully. At most, they should be warm to the touch.
  6. Seal beneath the bottom partial seal twice.

IMPORTANT: When sealing, press down hard on the sealer. This is the difference between a complete seal and a crummy, patchy one

Inoculating bags

However many bags you have to fill, scrape 1/5 as many plates into a sterile container. Inoculate with 1mL spore suspension

Inoculation day

Day before

Gather materials and load into van:

  • Boxes of S, M, L nitrile gloves
  • HandySteps
  • HandyStep tips and tip adapters
  • Six 500mL to 1000mL beakers for liquid inoculum
  • Field map of what needs to be inoculated, and with what
  • Three large autoclave bags for mixing sorghum

Autoclave the following:

  • Twenty squares of cheesecloth, wrapped in aluminum foil
  • One clean 2000mL beaker, foil on top
  • Three clean 250mL beakers, foil on top
  • Six 500mL bottles of diH2O

Scraping spores

It takes about 1 hour to scrape enough for 10,000 plants. Start early, move quickly.

Gather on a cart:

  • Two full spray bottles of 70% ethanol
  • Large stack of paper towels
  • Plates of S. turcica, in stacks of 10
  • Extra packs of sterile cheesecloth

Into the biosafety cabinet:

  • Two sterile 500mL beakers to collect spores
  • One sterile 2000mL beaker to hold all of the suspension
  • Ethanol lamp
  • Glass container with 95% ethanol for dipping L-rods
  • Sterile L-rods

Scraping procedure

  1. Spray your hands with ethanol after every time you grab something outside of the cabinet.
  2. Carefully open and unfold sterile cheesecloth and drape it over your 500mL beaker.
  3. Open plates and scrape in the biosafety cabinet. Pour a small splash (5-10mL) of cool, sterilized diH2O on the plate. Dip a glass L-rod in 95% ethanol and flame. Cool the hot L-rod in the water, then scrape the plate firmly.
  4. Every 10 plates, toss them out and grab new ones
  5. Every 20 plates, ethanol your hands and squeeze out the cheesecloth into the spore collection. Swap it out for a clean cheesecloth. Add the collected spore suspension to the largercontainer.
  6. Every 40 plates, check your progress with the hemacytometer

Using hemacytometer

Loading:

  1. Spray with ethanol and wipe dry with Kimwipe before use
  2. Mix the large inoculum container VERY well. Swirl both ways
  3. Add 10uL of spore suspension to each side. Carefully place the coverslip on, without sliding or smearing

Viewing under microscrope:

  • There are nine large squares, each bounded by triple lines, on each hemacytometer panel. Count spores only in the corner squares and center squares, not edge squares.You should be counting five large squares in total.See hemacytometer_usage.doc for instructions
  • If a spore straddles the top or left lines of a large square, count it. If it straddles the right or bottom lines, do not
  • Spray and wipe dry after use

Adjusting spore concentration:

  • Check spore concentration every 40 plates
  • Use to estimate how far along you are
  • Ex: If you have 250mL already, need 125mL of spores to make 3000mL of diluted spores, and need four 3000mL jugs of diluted spores, you’re halfway there
  • Add your spore counts to the hemacytometer_calculations.xlsx spreadsheet. Adjust the “final vol” cell to the required volume and then figure out how much spore suspension, water, and Tween 20 to add to make that volume
  • When you think you have enough, load and counthemacytometer twice (i.e., get four spore readings overall)
  • To add Tween 20, pour a very small amount into a small beaker and pipette it very, very slowly.

Inoculation day

Each person will sign up to complete either liquid or solid inoculum on a range. Liquid goes 2-4 times as fast as sorghum dropping.

Mix all bags of sorghum together in one large autoclave bag. This will limit spatial variation in inoculum “strength” and mitigate the effects of any inoculum bag with problems.

Set all HandySteps to 1 (i.e. 0.5mL per step)

One person will operate as a runner/gopher, getting everybody refills on liquid inoculum, etc.

1000mL of spore suspension will cover around 1800 plants.

One bag of sorghum (around 2000mL) will cover around 1000 plants.

Always bring extra! So much extra.