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OfflineSyncrip
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Registered: 10/24/20
Posts: 19
Last seen: 2 years, 7 months
Re: Pestalotiopsis microspora [Re: Bumbaclotjohnson]
    #27472446 - 09/18/21 12:10 AM (2 years, 7 months ago)

Apologies for necro-bumping, but I've spent the evening reading through this thread thinking about how to put together a proper selective media recipe for growing this organism. I figured it would be better to dump those thoughts here where the context is readily available, rather than have them floating around as their own isolated thread. As a disclaimer, my experience is more with molecular biology than microbiology/mycology, so if anyone with proper expertise with different media formulations wants to chime in it would be much appreciated!

Two posts from woodrow stuck out to me here:

Quote:

woodrow said:
An ideal selective medium would consist of a solution of mineral salts with with a nitrogen source and plastics added as a sole source of carbon. Bits of degraded plastic could be added to this medium in the hope of enriching for something that can grow on plastic. The medium would likely need to be incubated in the dark to prevent the growth of algae.




Quote:

woodrow said:
Agar is a carbon source for many organisms and microbes will consume the most readily available carbon source first and then move on to the more difficult. If you mix plastic and agar, the agar will be the first consumed and you will be selecting for organisms that break down agar but not necessarily plastic so agar plus plastic is a poor screening tek.




It seems like most of the attempts to grow P. microspora in this thread have either used media which either has another readily available carbon source (MEA + polyurethane), or doesn't have the trace minerals & nitrogen required for proper growth (plain water + polyurethane).

I started out just trying to source and price out all the reagents for the minimal media used in the original paper, minus the fancy Impranil DLF polyurethane. Here's what I came up with (prices in USD):

  • NaH2PO4 (Monosodium Phosphate) - $5.90
  • K2HPO4 (Dibasic Potassium Phosphate) - $65.83
  • (NH4)2SO4 (Ammonium Sulfate) - $9.49
  • MgSO4 (Magnesium Sulfate) - $9.99
  • FeCl3·6H2O (Ferric Chloride) - $13.95
  • ZnCl2 (Zinc Chloride) - $10.50
  • CoCl2·6H2O (Cobalt(II) Chloride Hexahydrate) - $66.80
  • Na2MoO4·6H2O (Sodium molybdate dihydrate) - $15.99
  • CaCl2·6H2O (Calcium Chloride Hexahydrate) - $13.63
  • CuCl2 (Copper(II) Chloride) - $12.99
  • MnCl2 (Manganese(II) Chloride) - $29.99
  • H3BO3 (Boric Acid) - $29.99
  • HCl (Hydrochloric Acid) - $10.45
  • Sodium Citrate - $11.95
  • Thiamine - $15.75
  • Tryptone (Replacement for Casamino Acids) - $34


TOTAL COST: $358.15 (plus taxes + shipping)

I swapped out the casamino acids for tryptone as it seems it's difficult to find casamino acids for sale outside of official scientific supply companies, which can be tricky to deal with and often won't sell to individuals. The Dibasic Potassium Phosphate seems to be the limiting factor here, as it's one of the more expensive reagents and at 33.5 mM you use up a good amount of it in the media recipe (about 5.8 grams per liter by my calculations). The cost of shipping so many different reagents, assuming you can't find them all in the same place, is sure to add up as well.

I started looking into some scientific supply companies thinking that it might be more easier to replace the NaH2PO4, K2HPO4, and sodium citrate with a simple pre-made phosphate buffer solution, and the slew of different metal salts with a pre-made solution. Teknova makes a 1000X "Trace Metals Mixture", that seems to cover all the bases pretty well. I was able to make an account on their site easily enough, and it looks like I don't need any special credentials to place an order.

Assuming you were able to get your hands on those reagents, a recipe for 1L of media might look like:

  • 50 ml 20X phosphate buffer
  • 1 ml 1000X trace mineral solution
  • 1 g (NH4)2SO4 (nitrogen source)
  • 0.5 g casamino acids/tryptone (amino acid source)
  • 0.03 g MgSO4 (don't see any magnesium in the 1000X mix)
  • 0.005 g thiamine (not sure why this was included in the recipe to begin with)


Any idea if phosphate-buffered saline would work in place of regular phosphate buffer? I know it's dirt cheap and very easy to get a hold of.

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InvisibleTawny

Registered: 09/11/21
Posts: 26
Re: Pestalotiopsis microspora [Re: Bumbaclotjohnson]
    #27473825 - 09/19/21 01:59 AM (2 years, 7 months ago)

[post deleted by user]

Edited by Tawny (09/28/21 12:29 AM)

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OfflinePTreeDish
Male

Registered: 04/22/18
Posts: 353
Last seen: 1 month, 3 days
Re: Pestalotiopsis microspora [Re: Bumbaclotjohnson]
    #27476303 - 09/20/21 09:51 PM (2 years, 7 months ago)


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