User:David Johnston Monje/Protocols

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Sterilization and microbial culturing

Corn/Teosinte Seed Surface Sterilization Protocol

  • Soak 20 seeds in dH2O for 12 hours (teosinte seeds should be soaked in 5% H2O2 instead to break dormancy)
  • Drain H2O and add Sunlight dish soap + water to the container and seeds and wash for 10 minutes in shaking water bath
  • Drain the soapy water and add 2.5% sodium hypochlorite to cover the seeds. Wash for 10 minutes.
  • Drain the bleach, and add 5% sodium hypchlorite to cover the seeds. Wash 5 minutes.
  • Drain the bleach and do 3 thirty second washes with distilled water. For teosinte, do an additional 5 minute wash with 70% ethanol to completely surface sterilize the seed.
  • To test for sterility, momentarily place the seeds on R2A media and culture that at 25 degrees for 10 days.
  • Germinate seed in the dark at 25 degrees for 7 days by placing in 25 x 100 mm culture tubes containing 10 mL of 6 g/L, pH 6 agar or 10 mL of well watered soil.

Corn/teosinte growth, surface sterilization, and endophyte isolation protocol

  • Sterilize 20 seed and germinate in culture tubes on water agar or soil.
  • After seeds germinate, place tubes in a controlled growth chamber, with an average relative humidity of 50%, under a light intensity of 100 µmol photons m-2s-1, provided by fluorescent and incandescent bulbs, and having a photoperiod of 16-hrs daylight and a 24ºC: 16ºC (light: dark) temperature cycle.
  • Observe plant growth for 1 to two weeks and harvest when plants reach the V2 growth stage (2 or three full leaves). Water agar grown plants will not be surfaced sterilized so proceed to step 11.
  • In sterile conditions, remove individual plants, gently wash off visible soil in tap water and place the plant in a 100 mL pyrex bottle. Add water and 1 mL of sunlight soap to cover the plant. Wash in shaker for 10 minutes.
  • Drain the water, and replace (covering the plant) with 2.5% bleach. Wash for 10 minutes.
  • Drain the water/bleach and add 5% bleach to cover the plant. Wash for 5 minutes.
  • Drain the bleach and do 3 thirty second washes with distilled water.
  • To ensure surface sterility, add 70% ethanol and conduct an additional 10 minute wash with 70% ethanol to completely surface sterilize the plant.
  • Finally, do one more 30 second wash with distilled water.
  • To test for sterility, momentarily place the plant roots on R2A media and culture that at 25 degrees for 10 days.
  • To isolate endophytes, thoroughly grind the plant (root, shoot, and embryonic seed) in an autoclaved mortar and pestle. Add 1 mL of sterile phosphate buffer to the homogenate and reserve 1 mL for DNA extraction (for TRFLP).
  • For culturing dilute the sample by taking 50 ul of juice from the homogenate, and add to 450 ul of sterile buffer in a eppendorf tube.
  • Serially dilute the sample by reisolating 50 ul of from the first tube, to a second containing 450 ul of sterile buffer, and then to a third to a final dilution of 1000 X.
  • Plate 100 ul of 1000X dilution on LGI, ½ PDA, and R2A (with cyclohexamide) filled petri dishes. Culture at 25 degrees for 10 days.

Carboxymethylcellulose(CMC)Agar Medium

  • 0.2% carboxymethylcellulose (CMC) sodium salt, 0.2% NaNO3, 0.1% K2HPO4, 0.1% MgSO4, 0.1% KCl, 0.05% peptone, 0.1% triton X-100 and 1.7% agar
  • This was adjusted to pH 6 then autoclaved and poured into 150 mm plates
  • To visualize cellulase activity, gram's iodine is flooded onto the plate and clear halos measured
  • Similar to that used in : http://www.springerlink.com/content/q7g54721205r26k3/fulltext.html

Pectin Agar Medium

  • 0.2%(w/v) of citrus pectin 0.2% NaNO3, 0.1% K2HPO4, 0.1% MgSO4, 0.1% KCl, 0.2% carboxymethylcellulose (CMC) sodium salt, 0.05% peptone, 0.1% triton X-100 and 1.7% agar
  • This was adjusted to pH 6 then autoclaved and poured into 150 mm plates
  • To visualize cellulase activity, gram's iodine is flooded onto the plate and clear halos measured

RNA rich Agar Medium

  • 1.5 g of torula yeast RNA was dissolved in 1 mL of 0.1 M PO at pH 8 and filter sterilized with a 0.22 um filter.
  • This sterilized RNA was added to 250 mL of autoclaved R2A agar media and poured into 150 mm plates.
  • After 5-7 days of microbial growth, plates were flooded with perchloric acid for 5 minutes and scored for clear halo production around colonies.
  • Ref: BARC Newsletter Issue No. 24, Founder's Day Special Issue 91. http://barc.gov.in/webpages/letter/2004/200410-13.pdf

Mineral Phosphate Solubilization Agar

Auxin production

  • R2A agar media was supplimented with L-tryptophan to a final concentration of 5 mM, then autoclaved and poured into 150 mm plates.
  • At day 4 the plates were overlaid with nitrocellulose cutouts allowing bacteria and their metabolites to infiltrate the paper
  • On day 5, nitrocellulose membranes were removed and treated with Salkowski reagent (0.01M ferric chloride in 35% perchloric acid) for 30 minutes and measured for reddish halos surrounding colonies days of microbial growth, plates were flooded with perchloric acid for 5 minutes and scored for clear halo production around colonies.
  • Ref: Appl Environ Microbiol. 1991 February; 57(2): 535–538. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16348419

Auxin production

  • R2A agar media was supplimented with 0.5% glucose, then autoclaved and poured into 150 mm plates.
  • At day 5 the plates were overlaid with 20 mL acetoin/diacetyl detection agar (75 g agar + 5 g creatine were autoclaved, cooled to 60 degrees, then mixed 3:1 with freshly prepared a-naphthol [75 g/L in 2.5 M sodium hydroxide])
  • After 30 minutes, plates are scored for red halos surrounding positive isolates
  • Ref: Journal of Basic Microbiology, Jan 2007, 34(4), Pages 277 - 280 http://www3.interscience.wiley.com/journal/114053778/abstract

Siderophore production

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