Lab Summary for 9/17/12

Discussed and viewed sexual and asexual life cycles videos for the protist - Phytophthora infestans, the Chytrid - Olpidium brassicae (causal organism of the club root pathogen), Saprolegnia, and  the Blastocladiales Allomyces (a really cool fungus).  

Baiting for Allomyces sp. from the soil surrounding the pecan pit in front of the Peterson building, utilizing non-viable heep seeds as the bait.  Observed zoospores (mitospores) from the initial microscopic examination.  Plated some of the water on YpSs agar plates.  Additionally, removed the heep seeds from the baited jar and placed the seeds into a small plastic petri dish with DS solution, parafilmed the petri dish and incubated for 48 hours at room temperature.  The initial YpSs agar plates were contaminated with bacteria and did not result in any fungal growth.  The heep seeds resulted in the amazing recovery of an Allomyces sp.  It was so incredible to see the zoospores (mitospores),  mitosporangia, meisporangia and rhizoids (photos below).  Transferred the fungal growth surrounding the heep seeds to YpSs agar plates in an attempt to recover a pure culture (update to come).  In addition, I took a video of the zoospores swimming in the petri dish (hopefully will be able to edit and post in the future).

Microscopic views of an Allomyces sp. 

A V8 agar plug of Phytophthora floating in soil water  was viewed microscopically to visualize the zoospores produced by the sporangia. I prepared a squash mount with a small piece of the agar plug stained with acid fucsion and observed via microscopic examination, coenocytic hypae, sporangia with zoospores present and  chlymadospores.

Briefly discussed Neurospora crasse crosses (mating) and will start those experiments next week (9/26/12).

Photo gallery update of the Riddell mounts from lab on 9/12/12

A. parasiticus

A. sojae

A. sojae

A. sojae

A. flavus

A. nidulans 

A. tamari

  

A. niger

A. oryzae

A. oryzae

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 . 

Lab Summary for 9/12/12

Seven different species of Aspergillus, A. niger, A. oryzae, A. sojae, A. tamari, A. nidulins, A. flavus, and A. parasiticus were available to observe microscopically, the differences in the morphology within the species.  Demonstrations of various ways to set up Riddell mounts were described.  Riddell mounts are utilized to examine fungi microscopically to minimalize the disturbance of various characteristic fungal structures, ie. conidiophores, vesicles, phialides, metulae, conidia.   

Riddell mount with agar block cut directly from A. flavus culture

Variant of Riddell mount with inoculated agar block and an agar block directly from A. niger culture

 Aspergillus sp. can be either bisereate and/or unisereate.

Selective media was discussed in order to isolate unknown fungi.  Water agar (encourages conidial growth, lacking nutients), Rose Bengal agar (has antimicrobial properties), Potato Dextrose agar (PDA), Cornmeal Agar (CMA), 1/2 strenght PDA (encourages conidial growth while providing some nutrients)+ Ampicillin (to discourage bacterial growth).

Sub-culturing was briefly discussed.

Lab Summary for 9/5/12

At the beginning of class, we reviewed everyone's blog posts.   Then we moved onto practicing how to identify fungi from culture.  Several fungal cultures were available for microscopic glass slide mounts: Aspergillus niger, Pythium ultimum, Thielaviopsis bassicola, Altenaria brassicicola, and a Cladisporium sp.  We used two different techniques, squash mounts and tape mounts, to visualize them under the compound microscope.   A squash mount is used with a drop of water on the glass slide, a small section of the fungus from the agar plate and squashed with a glass cover slip.  A tape mount was also used with a drop of water and a piece of tape to lift the fungal structures.  The squash method was a better method in order to see the fungal structures, such as conidiophores, hyphae, and spores to aid in identification.

Aspergillus niger: Observed only pigmented single celled spores using the tape mount method.  Observed septated hypae, condiophores, and spores using the squash mount.  Tried several squash mounts to try to observe the phialides, but was unable to obtain a good mount.

Pythium ultimum: Observed coenocytic (lacking septa) hyphae, with sporangia characteristic of Pythium sp. Causes root rot on various hosts such as cool season grasses and ornamentals.

Thielaviopsis bassicola: Observed phialides conidiophores, and aleuriospores characteristic of Thielaviopsis sp. Causes a plant disease commonly referred to as black root rot.

Altenaria brassicicola:  Observed pigmented conidiophores with chains of pigmented conidia with cross and longitudinal septa in chains. Septated hyphae.  Can be a plant parasite, causing leaf spots and but can also be saprophytic.

Cladisporium sp: Observed pigmented, dark conidiophores with one and two celled conidia some in chains. Can be plant parasitic on plants such as spinach causing leaf spots.  Can also be saprophytic.

Hemacytometer

We were instructed via a You Tube video, on how to use a hemacytometer and counted Aspergillus niger spores in a 1ml water suspension.  100ul of the spore suspension was placed under the cover slip and 5 squares were counted and averaged.  My spore count calculated into 2.9 x 106/ml.

Square grids of hemacytometer as seen under the compound microscope.