Agar culture

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John
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Agar culture

Post by John » Sun Dec 10, 2017 1:27 pm

Some agar recipes from the excellent article below by Edward F. Haskins

1. 0.75% Water Agar (.75 WA)
Bacto agar 7.5 g Glass distilled water 1 L

This agar has been successfully used to germinate a number of Myxomycete taxa.
After germination of spores, transfer the amoebae and swarm cells to other types of agar media (e.g. 1.5% WA, wMY or CM/2 agar) along with appropriate food microorganism(s).

2. 1.5% Water Agar (1.5 WA)
Bacto agar 15 g Glass distilled water 1 L

3. Half Strength Bacto Corn Meal Agar (CM/2)
Bacto corn meal agar 8.5 g Bacto agar 12.5 g Glass distilled water 1 L

4. Scratch Corn Meal Agar (SCM)
Albers yellow corn meal 20 g Difco agar 17 g Glass distilled water 1 L

Useful methods
https://www.google.co.uk/url?q=https:// ... KslGdIaaUM
Edward F. Haskins 5 Professor Emeritus Department of Biology Box 351800 University of Washington


Cultivation from spores. From the article above.
If sufficient spore material is available, set up germination
cultures on 0.75% WA, 1.5% WA, wMY, and CM/2 agar (see Formulae of Media). If there is a
paucity of material, try 0.75% WA first; then if no germination occurs, 1.5% WA, wMY or
CM/2 agar. Use a fine marking pen to divide the bottom of the petri dish into quadrants. Draw
small circles in each quadrant which will serve as areas for inoculation of spores. These circles
will allow you to quickly locate the deposited spores as you check at intervals for germination.
9
I prefer to use an alcohol flamed #5 jewelers forceps to pick up spore material and inoculate
the surface of the agar in each of the quadrants using a gentle slashing motion to make sure some
of the spores are submerged and others are left on the surface. Other workers use a sterile
transfer needle to pick up a small sample of spores to spread on a germination medium. Some
workers heat the needle, plunge it into an agar plate, thus coating the tip with agar which makes
the end of the needle conducive for picking up spores. In both methods, individual spores should
be well separated on the substratum. Germination usually takes 12-36 hours but may in some
species occur within a few hours.
Spore germination can be observed by inverting the agar plate on the stage of a compound
microscope from which the clips or mechanical stage have been removed. Use a 5X or 10X
objective with 10X or 15X oculars to view the spore, amoebal and plasmodial phases at 50X
150X. Typically a dissecting microscope provides less resolution at a comparable magnification.

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John
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Re: Agar culture

Post by John » Fri Jan 12, 2018 2:57 pm

I prepared some 0.75% water agar plates under sterile conditions by dissolving 0.75g of agar in 100mL of distilled water, sterilising in a pressure cooker for 30mins and then pouring around 10-15mL per sterile 90mm petri dish.

Once prepared, they were used in non-sterile conditions and seem quite resistant to contamination with microorganisms in the short term (probably due to the low levels of available nutrients).

A small amount of sclerotia on filter paper was added to the centre of two plates along with a non-sterile oat nearby (sterile oats are recommended to reduce contaminants).

A plasmodium appeared and grew quickly on one plate (the other piece of sclerotia was unsuccessful). Below is a photo taken after 12 hours.
20180112_105850.jpg

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Re: Agar culture

Post by John » Sun Jan 28, 2018 3:48 pm

See also this very useful guide by Fred Spiegel

http://slimemold.uark.edu/pdfs/usefulmethods.pdf

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