Germinating spores - observation by the hanging drop method

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John
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Germinating spores - observation by the hanging drop method

Post by John » Fri Jun 30, 2017 7:41 pm

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This is a way to observe spore germination under the microscope.

Needed: microscope slide with cavity well, cover slip, petroleum jelly.

1). Put an even thin layer of vaseline arond 3cm square on your left palm. Pick up a cover slip with the right hand and rub one edge of the cover slip against the left hand. This allows the vaseline to build up on one side in an even layer. Repeat on all four sides.

2) Place the cover slip on a flat surface (vaselined side up)and add a very small drop of spore suspension to the centre. Now pick up the cover slip and quickly invert it so drop remains in the centre of the cover slip.

3) Place the cover slip (with drop on the underside) on top of the slide over the cavity.

4) Observe using over 10x - 40x objectives.

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John
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Re: Germinating spores

Post by John » Sun Dec 17, 2017 3:53 pm

I found that hanging drops were very prone to drying out quickly and therefore attempted to germinate spores in a vial of distilled water and transfer to a hanging drop for observation.

A fruiting Physarum polycephalum culture gave me some fresh spores to work with.

1) A couple of ml of distilled water was added to a clean glass vial. A couple of sporangia were crushed over the water using sterile forceps and spores transferred by repeatedly dipping the forceps in the water.
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2) A hanging drop slide was prepared and examined at x10 - x40. No germination was observed after 2-3 hours.

3) Another slide was prepared from the vial at 24 and 48 hours and spore germination was observed :) Swarm cells with flagella were seen moving freely around and rotating rapidly.
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Tips: best observed with air objectives and a microscope equiped with DIC. The use of oil immersion objectives is difficult with this method as the cover slip is mounted on a layer of vaseline and is easily squashed. I think it is better to prepare a couple of mls of water/spore mix and prepare hanging drops daily as they dry out very quickly. Prepare the hanging drop with the smallest amount of liquid possible as this will give you a thin layer and make observation easier.

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John
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Re: Germinating spores

Post by John » Sun Jan 28, 2018 3:56 pm

Drops of the germinating spore suspension were transferred to sterile 0.75% water agar. After 2-3 weeks, very small yellow plasmodia could be seen under a stereomicroscope.

At 4 weeks, small oat flakes were placed nearby in order to encourage growth of plasmodia.

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