Trying to calculate a germination percentage of fern spores grown in sterile media and counted under a microscope. Overlapping spores makes counting difficult. However cultures must remain sterile.
Dear Sam,
I remember that a beloved and ingenious Venezuelan colleague named Teresita, who did the same thing as you, came up with the method of planting bacteria. Making the "movement in S" serial, with the typical instrument of plaque bacteria, managed to disperse the spores not only uniformly in the plate, but it considerably reduced the over position.
Surely it will work for you too!
Hi
1. Obtain a spore suspension from the fungus in question and then serially dilute. 2. At the last dilution you should measure the concentration with the Neubauer chamber (number of spores / mL).
3. place 1mL of the lowest dilution on a plate with a very solid water-agar medium (20g / L).
4. Monitor spore germination every 4 hours. Depending on the type of spore it can germinate in 4 hours or more.
5. To monitor the germination of the spores, place a disinfested microscope with alcohol 70 into the laminar flow chamber (so that there is an aseptic environment),
6. Open the plate where the spores were sown and observe under the microscope if the germination began.
7. If any spores are already germinated, transfer them to another desirable culture medium.
8. To transfer it is sufficient to cut the agar-water medium around the germinated spore so that you get a small piece of the medium containing the spore and place the part where the spore is in contact with the desired culture medium. So you will have a monosporic culture in a few days.
Hi Sam,
I had the same problem in my undergraduate thesis (http://132.248.9.195/ptd2008/agosto/0630285/Index.html ), with spores of Cyathea, Alsophila and Phlebodium, until I solved it through this protocol:
Protocol for the disinfection of fern spores:
1.- Sieve the spores in mesh # 200 (74 μm).
2.- Place 0.01g of spores in 1.5ml centrifuge tubes.
3.- Add the disinfectant solution (0.09% NaOCl added with tween20).
4.- Shake vigorously to ensure the penetration of the disinfectant agent on the surface of the spores (15 to 30 minutes).
5.- Centrifuge at a speed of 1000 rpm for 5 minutes.
6.- Subsequently take the tubes to the laminar flow hood and rinse inside it.
7.- The rinse is done by discarding the supernatant and adding, with the help of a micropipette (with sterile tips), sterile distilled water.
8.- After each rinse step 5 is repeated; and after 5 rinses the spores will be resuspended in a volume of sterile water and mixed to obtain a homogeneous spore solution.
9.- The desired volume (60-120μl) is taken from the solution and sown in containers with previously sterilized culture medium.
10.- Once grown, close the container tightly to avoid
contamination and placed in an incubation room.
Steps 3 and 4 are critical, check the concentrations and times, because depending on the species, spores can be damaged.
goog luck
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