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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

• 10 g/L glucose + 0.373 g/L NH4NO3 + 0.41 g/L MgSO4 + 0.295 g/L NaCL + 0.003 FeCL3 + 0.7 g/L CaHPO4 + 20 g/L agar
• This was adjusted to pH 6 then autoclaved and poured into 150 mm plates
• After 5 days growth at 25 degrees, clear halos were measured
• Reference : http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-41PNYNT-7&_user=1067211&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000051237&_version=1&_urlVersion=0&_userid=1067211&md5=1ba0dfc48ab1c3e9a86a73ab85e16484

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

• Microbes were grown on normal R2A agar media for 5 days at 25 degrees.
• At day 5 the plates were overlaid with 30 mL O-CAS overlay
• O-CAS overlay was made by mixing Chrome azurol S (CAS) 60.5 mg, hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg, Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 30.24 g, 1 mM FeCl3 · 6H2O in 10 mM HCl 10 mL and 2% Agar.
• After 15 minutes, a change in color will be observed in the overlaid medium, exclusively surrounding producer microorganisms, from blue to purple (as described in the traditional CAS assay for siderophores of the catechol type) or from blue to orange (as reported for microorganisms that produce hydroxamates).
• Ref: Journal of Microbiological Methods Volume 70, Issue 1, July 2007, Pages 127-131
• http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-4NGRRNR-1&_user=1067211&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000051237&_version=1&_urlVersion=0&_userid=1067211&md5=e3666005a65ed298d3d5733749ff485e