Unknown fungal project :    "What is wrong with my tree?"

Unknown #1 
Source: Oak tree branch from home owner in Travis county Texas

How to process branch tissue for fungal isolation 

 1.   Scrub branch with bacterial liquid soap in running water to remove lichen, mosses, and mildew that  

       occur on surface.

 2.   Surface sterilize the sample with alcohol and flame.  

 3.   Remove bark and wood to cambium layer with sterile chisel.  

 4.   Sterilize chisel and remove strips of tissue or quarter the smaller branches.  

 5.   Cut angular samples from the strips with sterilize pruner. 

    6.   Place chips into beaker.

    7.   Add 20% bleach solution to the beaker until the chips become submersed, swirl the solution over

          chips for a few seconds.  Time for 3 minutes.

    8.   Decant bleach.  Add sterile distilled water into beaker until chips are submersed, swirl water then

          decant water. 

    9.   Pour more sterile distilled water over the chips until submerged and time for 1 min.  Decant water.

  10.   Pour the chips onto sterile paper towels. 
  11.   With sterile forceps, place 4-5 chips on APDA+ media. (Recipe for media: 500ml distilled water,   

          10grams dextrose, 5 grams instant mashed potatoes, 6 grams bacto agar.  Autoclave for 20 min. 

          Allow agar to cool to 50 degrees and add 500ul sterile lactic acid.  Pour into sterile petri dish)

  12.   Place all plates into zipper  bag and date with sample number on the outside of the plastic bag.   

          Place in 25 degree incubator.

  13.   Check plates periodically for 5-21 days for fungal growth.

Initial plating of branch wood chips

Beginning stages of fungal growth 7 days  from plating wood chips

Transfer of the fungal growth, resulting in a pure culture of fungus

Lactophenol blue stained compound microscopic view of
condiophores with released chains of conidia typical of  Ceratocystis fagacearum, the causal agent of oak wilt .