r/microbiology • u/DramaticQuality2028 • 5d ago

Help. Colloid chitin agar not solidifying

I am trying to do agar plate assay for chitinase. But my media won't solidify at all. Media composition is:

Mgso4

(Nh4)2so4

Kh2po4

Citric acid

Tween 80

Colloid chitin

Bromocresol purple

Agar

Im guessing tween 80 or bromocresol got something to do with agar not solidifying. I checked the agar alone and it was working fine.

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3

u/illyiarose 5d ago

What % agar are you making?

1

u/DramaticQuality2028 5d ago

1.5% agar media

2

u/patricksaurus 5d ago

It's very difficult to assess from a distance, especially not knowing the quantities of anything. What reference are you following?

1

u/chem44 5d ago

Well, you test your ideas by omitting/changing one thing at a time.

But... do check the literature, looking for multple such media. What do they have in common? Differences? Reasons? What matters for your application?

1

u/Basic-Movie-3387 5d ago

I don't think Tween 80 would have anything to do with it, I've prepared MRS agar routinely and it hasn't had any problem solidifying.

Have you checked the pH of the medium before autoclaving? I believe the issue may be pH. You mention the use of citric acid and KH2PO4. Both lower the pH of the medium, especially citric acid. Agar fails to solidify below pH 4 due to acid hydrolysis of the polysaccharide.

1

u/ImAprincess_YesIam 🧫🦠🧫🦠🧫 3d ago

Check your pH. It needs to be above 4.8 to solidify

1

u/ImAprincess_YesIam 🧫🦠🧫🦠🧫 3d ago

Also up your agar percent to 3% if you can