Obtain a flat leaf from the Magnolia grandiflora alpha tree located behind Winston Hall.
-Choose a leaf that is relatively disease- and damage-free.
-Look on underside of leaf for insect egg cases
-Note compass point. Generally, use a leaf from the South or the North.
-The best would be one leaf from each side on one particular day
-Note the height of leaf sampled from the ground in meters.
Cut off the stem, right at the edge of the leaf blade
Rinse the leaf in running tap water. Dry well by blotting with paper towels.
If you have a phone that can take pictures and can download or transmit a jpg file:
-Take a BLACK and WHITE photo.
-Place leaf on 1 cm square graph paper or similar size reference, on top of white light box.
-Cover (gently!) with glass plate to flatten as much as possible
-Goal is to have high contrast photo with visible size standard
-Use photo to calculate surface area of leaf using ImageJ
-You can include in the frame the sample name
-Written legibly on an index card, etc.
-Take a COLOR photo
-Leaf does not have to be flat.
-Include a color reference in the photograph.
-BOTH photographs should be renamed with “DateUserName” – see next.
On an Endophyte Record Form record the name of the sample:
-“Dateusername” – YYYYMMDDuseramea I.e., the date and your username and an additional letter if multiple samples. Ex.: 20140130taguebwA
Record the specimen name ( grandiflora alpha, leaf), date, compass point, height, time of day, weather, other special notes
Weigh the specimen and record weight in grams.
Use the appropriate cork borer to produce leaf disks.
-CURRENTLY USING 2.5 mm radius borer
-Put leaf on PLASTIC “cutting board”
-Press down gently on the tissue with a slight circular motion
-Don’t press too hard – it will damage the cork borer
-Disks that stay with the leaf can be gently “worked” off
-Avoid using diseased or damaged leaf tissue
-Avoid central vein.
-We are currently generating >= 80 squares. More is better!?
-Collect them on the cutting board or into container.
Place leaf disks into a clean small glass container (beaker, petri dish, etc).
-From here on, use care while handling the leaf disks.
-Leaf disks should only be contacted by the container, the sterilizing solutions or flamed, sterile forceps.
-Do this in the hood if possible.
Cover leaf disks with 95% ethanol.
-Agitate petri dish for 10 seconds then drain into a sink or (better) into recycling container for ethanol.
-Sterile forceps can be used to hold back leaf disks.
Cover the leaf disks with 10% bleach (0.6% hypochlorite) and agitate for 2 minutes.
-Drain bleach into waste container.
Cover the leaf disks with 70% ethanol, agitate for 2 minutes.
-Drain again
In the hood, carefully remove disks to clean sterile petri dish, allow them to dry well before plating.
Carefully place leaf disks onto culture media.
-Flamed, sterile forceps usually work well for moving leaf squares.
-Use “PDA-quad” petri dishes
-Place one circle in each quad.
Plates should be labelled on the bottom with sample name, other info.
-Wrap plates individually with micropore tape. Put plates into storage cabinet.
Clean up area. Place equipment back into boxes. Replace benchtop paper if necessary.
Check daily over next 5 days to 2 weeks from the emergence of fungi from leaf disks (usually 7 – 10 days).
Score emergence of fungi before fungi overgrow each other.
-Score as follows: Number of disks with fungi vs. number of disks without
-For each disk, lists number and morphotypes.
(Additional) Score after additional growth/Move a “0 fungi square” alone to a new petri dish with the same media, rewrap and score for late emerging fungi/Isolate for pure culture
Record any other pertinent notes on Endophyte Record Form. If scoring fungi over a number of days, note PAGE (1/3, 2/3, 3/3).
Dispose of plates in biohazard bag in clear Lucite container. Make a note on Endophyte form that experiment is terminated; include date terminated.
Check daily 4 days to 2 weeks after plating for the emergence of fungi from leaf disks (usually 7 – 10 days). Days of growth to be standardized? If you have a cell phone camera, consider ~daily photographs?
Score emergence of fungi before overgrowth on Endophyte Record Form-Fungi to score will be associated with a leaf square.
-Contamination can be ignored if it does not interfere with scoring of fungi numbers.
-Record the following for each dish:
•Media used
•Fungi present in a leaf disk (regardless of number)
⇒i.e., If 3 show fungi and 3 do not, record “3+, 3-“
•Record number of “rings” emerging from each leaf square
⇒ i.e., total number of fungi per leaf disk
⇒i.e., 3, 1, 1, 0, 0, 0
•Record species or morphtypes of fungi present
⇒i.e., 3 ghost (fast), 1 pink, 1 white (slow)It is better to be a “lumper” than a “splitter.
•Consider color, rate of growth, color underneath, more? We have seen three distinct, common morphotypes.
⇒Pink (fast growing, relatively common, easy to observe)
⇒Ghost/white/translucent (fast growing, common, may include multiple species?, more opaque with age/conidia?)
⇒White/opaque (slow growing, common, may include multiple species?)
⇒RARER: Black, orange, blue/metallic, green, pasty
(Extra) If instructed, move a “0 fungi square” alone to a new petri dish with the same media, rewrap and score for late emerging fungi. If instructed, isolated fungi for pure culture and long term storage.
Record any other pertinent notes on Endophyte Record Form. If scoring fungi over a number of days, note PAGE (1/3, 2/3, 3/3, etc.).
Dispose of plates in orange biohazard bag in clear Lucite container. Make a note on Endophyte Form that experiment is terminated, include dateScore emergence of fungi before overgrowth on Endophyte Record Form(Extra) If instructed, move a “0 fungi square” alone to a new petri dish with the same media, rewrap and score for late emerging fungi. If instructed, isolated fungi for pure culture and long term storage.
GENERAL: Clean is good!
Soap is good.
-Washing your hands, wrists and forearms often with anti-bacterial soap is a good habit.
-Anything that does in the hood should be clean! So don’t put your head in the hood!
Ethanol is good.
-In the hood: Wipe down the benchtop inside, hood “lip”, inside surfaces of hood with 70-95% ethanol, before use. Repeat as necessary
-Petri dishes: Before opening ANY dish (fresh from the refrigerator or already growing cultures), give them a gentle wipe with an ethanol-dampened Kimwipe or paper towel. Be careful NOT to remove the writing on the bottom of the dish!
-Equipment: wipe down or place in ethanol for surface sterilization. This includes container for leaf disk sterilization, before starting
Flame is good.
-Flaming metal utensils (blades, forceps, scissors) often helps!
-Put utensil in beaker with ethanol. Remove. Pass through flame of alcohol burning. Let flame go out; pause a moment to let metal cool. Move tissue. BE CAREFUL: Do not put flaming metal back into ethanol!
Cover is good.
-Work under the hood, if possible.
-Keep material covered, especially during sterilization.
-Cover with glass or plastic petri lid, not paper towels or Kimwipes
-Only open petri dishes to move tissue, do not leave lid off.
Clean is good.
-Clean up after yourself. Throw away bits of parafilm, bits of plant material.
-Put tools back into appropriate bins.
-Finished with the hood? Clean it up, organize, give it a dose of ethanol.
-Replaced soiled or green-stained bench top paper
-Remove clutter, wipe down counters and lab tops
Isolating Fungi into a Fresh Culture
Select a single type of fungi to isolate. It should be far enough away from other cultures as to not be touching other types of fungi that could potentially contaminate the selected culture.
Sterilize the area that you will be working in under the hood with ethanol and a kimwipe.
Dip the ends of a pair of forceps in ethanol and run through the flame of an ethanol burner. Allow flame to burn out itself for complete sterilization before moving on. This should happen fairly quickly.
With the newly sterilized forceps, quickly remove the top of the agar plate (as to not expose it to potential contamination for too long) and pinch a tiny edge off of the perimeter of the culture which you are isolating. Replace the lid.
Lightly tap the tip of the closed forceps to the center of a fresh agar plate and immediately close the lid.
Check in on your newly isolated culture periodically to observe its growth. Growth should begin within 24 hours and will continue to occur rapidly.
