if it necessary to use agar media at such low pH , then try to prepare your medium at double strength and the agar at double strength as well, and sterilized them separately and mix them just before use

Try putting agar at double concentration

I will try that too but the reason is hydrolysis of agar which occurs at that pH during sterilization.

Thank you @ Said Amrani for your contribution. But I am trying to make pH gradient plates where I need to pour over another medium which is at pH 6.5.

How about adding calculated amount of 1% citric acid after autoclaving media at normal pH? Do you think that will work?

I have an old school suggestion - try making gelatin plates. See if they survive the autoclaving at pH 3. If you are culturing environmental samples, you can incubate at lower temperatures such that gelatin does not melt.

My advice: avoid agar for low pH experiments. Even if it solidifies, growth-inhibitory concentrations of weak organic acids may be released due to (partial) hydrolysis. Electophoresis-grade agarose is already better. Autoclave twice concentrated medium and agarose separately, cool to just above melting point, mix and poor plates. Floating filters/gelatin/silica may be better but no experience with those.

if it necessary to use agar media at such low pH , then try to prepare your medium at double strength and the agar at double strength as well, and sterilized them separately and mix them just before use

This is better Han what I suggested it should work

I second that. I will keep you all posted how it goes.

sterile citric acid or tartaric acid 3% solution added aseptically in 0.35 ml quantity to a plate while pouring sterile agar medium of pH 6.8 or 7.0. Please check the Laboratory Manual for Microbiology ... Harrigan. This medium is generally used for molds isolation from soil , water or other sources.

I used to prepared medium at double agar strength and imediately affter pouring i put the plates in the refrigerator. the maiority of the timmes it worked!

The double strength method sounds promising. Have you considered Malt Agar? Perhaps some help can be found in the DIFCO manual:

http://www.bd.com/ds/technicalCenter/misc/difcobblmanual_2nded_lowres.pdf

Good Luck.

Not sure if you were ever successful. Just saw this question. I agree, with Lesley, use Gelrite (autoclave separately to your nutrient broth medium you want), make sure there are divalent cations in the latter (Mg or Ca work well) and mix right before pouring. You have to be really fast and pour/pipette immediately or it will set. Use at 0.8-1% or up to 1.2% depending on how "solid" you want it. I think agarose will also work, and even the agar autoclaved separately from the acid sort of works. I doubt gelatin will work though. Gelrite gives amazingly good "solid" plates at low pH.

Agar can for sure solidify at pH 3.0 (lowest I tested). Problem is in fact that agar hydrolyzes or/and is modified during autoclaving at pH3.0 and therefore unable to solidify anymore. From this cause, it is crucial to adjust the pH of the agar medium after autoclaving. Simplest way to do that and keep medium sterile is to prepare two same volumes of the agar medium and to use one to estimate HCl needed to lower pH (unsterile). Same volume of HCl is simply added to the another volume under sterile conditions.

you should do two solutions: i) water agar half the final volume and ii) with double salts concentration and pH 3, sterilized separately and at a suitable temperature quickly mix and pour into plates

@ Branislav, @ Maria-Isabel, thanks all of you for your contribution. I had been adding precalculated amount of acids to sterilized media right before pouring, however finding right temperature of mixing (not too hot and not too cold) was difficult especially when I had to add more volume of acid (or more concentrated one). Instead, I have been making double the concentration of agar and media separately and mixing them together at the time of pouring, so far it's been working great for me.

Hey guys I have made these plates at double concentration at low pH's. As it is double will this affect the amount of growth i.e. Very heavy growth as there is double concentration and therefore double the amount of nutrients? 

Thanks 

@Chris, sorry but it looks like my explanation was not very clear. When you mix media and agar your volume getting doubled at the end so the actual concentration of media would be same as your standard media.

Hi!

During autoclaving  at pH 3.0 agar is changed so that can not polymerize anymore! Therefore you have first to autoclave your media and adjust pH afterwards. How to do it and keep sterility? You have to work in parallel with two equal volumes of media. One volume you will use to adjust pH under normal (unsterile) conditions and discard after that. Same amount of HCl needed to lower pH to 3.0 you will add to the second volume under sterile conditions. Works very well. Good luck!

Dear Bikram,

I make 2 fold concentrated media with the required pH (2, 3 and 4) and sterilize it separately. Parallely I sterilize 40 g/L agar in the 1X DI water and sterilize it separately. After autoclaving I cool both of them and mix together and pour plates. I didn't had any problem. Agar plates solidify very well.

Don't autoclave agar at acidic pH.  At low pH and after autoclaving agar looses its capability of solidifying.

I hope this will solve your problem. If you have any questions please don't hesitate to contact me.

Hello Bikram

I am working on fungal species and interested in measuring their growth at different pH. I am using malt agar media for this. Can you please tell me how can i assign a particular pH to my plates.. i mean which acid or base you used for your studies and how you added that to the media..?

Dear Navjot, I would encourage you to go through different options mentioned in the post already. TLDR; either you can add pre-calculated amount of acids into the media (after heat treatment at normal pH) or make 2X media (at desired pH level) and 2X agar separately and mix them up later before pouring your plates. Thank you.

Hi Navjot Kaur

I'm doing a similar experiment with a fusarium species on pH 4, 5, 6, 7, and 8 PDA plates. What I did was to prepare the PDA for pH 4 with 90% of the water required, and the last 10% of water is amended with enough acid to drop the final pH to 4 (this was predetermined previously by adding acid to the PDA solution and recording how much acid I needed to reach pH 4). The 90% water PDA and 10% acid solution were autoclaved separately.

I ran into some problems though. My control 2 (same steps above, without acid) and my control 1 (no amendment added, PDA prepared normally) had significantly different results. Not too sure why that was the case. Another issue was the choice of acid. I used acetic acid, and I discovered it may have a significant antimicrobial property beyond acidity. So I would suggest you use an inorganic acid like HCl.

Let me know how your experiment went.

Hi vikesh ajith

Thanks for your suggestion..just need to know something, did you use any buffers to keep the pH constant throughout your studies?

Hi Guys,

Should we use buffers or acids/bases to see the effect of pH on bacterial growth? I am working on Lactobacillus delbrueckii .

Thanks for your suggestions in advance.

Navjot Kaur from what I read ideally you should use a buffer, especially if it's a long experiment and you are comparing between two different fungi (they might affect the surrounding pH at different rates?) Check out this paper:

https://www.jstor.org/stable/3758289?seq=1#page_scan_tab_contents

I did not use a buffer the first time around, though I'll probably use a buffer next time.

Dear Vikesh,

Thanks a lot for your suggestion and sharing the paper.

Actually,I have been using buffers but after reading lots of answers/suggestions from the research gate I realized that if I am using an acidic buffer then actually I am supplying the medium with many more ions or salt that can be used as nutrients than the addition of just HCl.

Vinita Sharma yeah, I've read that too. I heard there are some non-nutritive buffers that the fungi would hopefully not consume, if you really need to use a buffer.

Vikesh Ajith thanks a lot.

Thanks a lot Vikesh. I will try to find non-nutritive buffers.

Hi

I want to make 2% of the malt extract media which I usually make using malt extract and agar in 500 ml DI water. I want to bring the pH to 5 using 0.1M sodium acetate and 0.1M acetic acid buffer (making 200ml). I want to know if I should make my media in 300 ml DI water and then pour the buffer in it, adjust the pH and then autoclave or I should prepare the media in buffer only, adjust the pH and then autoclave.

Navjot Kaur; for malt extract media, I would suspend media in distilled water and or buffer (per your need), dissolve, adjust pH, and sterilize by autoclaving. I would not re-heat or autoclave media at lower pH (~3.5) but pH 5.0 should be okay to autoclave. Good luck.