Do you really need to adjust pH of medium prepared from powder? - (Aug/08/2007 )

Hi there,

I've been using liquid media directly purchased from manufacturer. I'd like to start saving cost and am planning to switch to powder form, in which you only have to add sodium bicarbonate and ultrapure H2O, then filter-sterilize. I've read the method of preparation and there is a step in which you have to adjust pH.

So, I was wondering if there's anyone out there who had been preparing powdered media into liquid and skipped the pH adjustment and are doing fine... Wouldn't phenol red already sufficient to pin-point the range of the pH? Would plus/minus a value of 2 in the pH of media affect the cell, just as long as the phenol red is not pink (above pH8.1) or yellow (below pH 6.6) but orangy in color?

Well, not that I'm lazy to adjust the pH, my lab don't have a working pH meter for quite a while... and I've been using pre-prepared buffer like Tris-Cl purchased directly from companies. It's not cost saving at all... so, working with powdered cell media is a start.

Many thanks for your reply.

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Hi!

I don't know what types of cell you're using, me myself handles only bacteria and fungi, so perhaps the answer is only valid for those...

In my lab we're using premade medium-mixes (in powder form), and I've never adjusted pH on that... I think they already contain buffers that keep the pH in the right range.
But when do you add sodium bicarbonate to the media would change the pH, wouldn't it?

We usually use media from Difco.

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You should not play easy with pH values like 2 points up or lower, it may be fatal to your cells. As a rule, It is important to obtain pH value near 7.4
Here some words about this


Most cell lines proliferate in medium with a pH of 7.4, and they exhibit decreased viability and rates of proliferation as the medium becomes progressively more acidic or more basic (Eagle, 1973). Culture media must buffer the CO2 and lactic acid produced as cells metabolize glucose and glutamine. Historically, bicarbonate, HCO3, in conjunction with atmospheric CO2, has been used as a buffering system. Each basal medium formulation has a recommended concentration for sodium bicarbonate, usually 20 to 40 mM, to maintain pH and osmolarity. Media that are to be incubated in an elevated CO2 atmosphere contain higher concentrations of bicarbonate than those designed to be used at ambient CO2 levels. Most cell culture media require an atmosphere of 5% CO2 to maintain pH 7.4. However, certain media contain levels of bicarbonate that require different amounts of CO2. For example, DMEM containing 3700 mg/liter of sodium bicarbonate equilibrates to ~pH 7.6 in a 5% CO2 environment and requires 10% CO2 to maintain pH 7.4. The low pKa of bicarbonate (pKa = 6.1) makes it a poor buffer around pH 7.4, and, in the absence of atmospheric CO2, the breakdown of H2CO3 formed from bicarbonate releases CO2 that comes out of solution, causing a rise in pH. With the development of Good buffers (Good et al., 1966), nontoxic buffering agents effective in the pH range of 6 to 8, such as PIPES (pKa = 6.8), MOPS (pKa = 7.2), TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; pKa = 7.5), and HEPES (pKa = 7.55), became available to the research community. HEPES in a concentration range of 10 mM to 25 mM has become a standard buffer in serum-free medium, but it is used in addition to and not in place of the bicarbonate and CO2 system. Phenol red is an indicator dye that is commonly added to medium to provide a visual assessment of pH. Red at pH 7.4, it becomes orange (pH 7.0) and then yellow (pH 6.5) as the pH decreases; it turns violet (pH 7.6) and purple (pH 7.8) as the pH rises. Culture medium should generally be replaced as the phenol red changes from orange to yellow, which reflects the accumulation of lactic acid.

Materials

Powdered medium without NaHCO3 or HEPES
HEPES (e.g., Research Organics)
NaHCO3 (e.g., J.T. Baker)

1. Dissolve powdered medium in water with gentle stirring.

2. Add HEPES (mol. wt. 238.3) to give a final concentration of 15 mM, and stir until dissolved.

3. Add NaHCO3 to the recommended concentration for the basal nutrient medium being used and stir.

4. Add other medium components, and adjust the pH to 7.4


If you have temporary problem with Ph adjustment use pH test Strips 6.0-7.7 Sigma p-3536

May be I don't understand correctly, but don't adjust pH with Tris-HCl only with NaOH

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pH can also be very important for certain exp, e.g. viral infection of cells, 0.2 point different could cause a huge shift in the efficiency. I would really suggest that you should pH your medium. Cells are a bit more fussy than bacteria and fungi. Even if you only want to grow cells, you still should be careful when preparing your medium so that you get less chance for problem in your experiments later (at least to make sure that your observed results are not due to any medium-related effects).

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In my old lab, our tech used to make DMEM and she always checked the pH just to be absolutely sure.

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Hi circlepoint. The excerpt is very useful and thanks also for including the method of preparing the media. I'll work it out somehow with the pH meter (or other pH indicator).

Hah. Apart from saving cost, another reason I'm changing to powder is the space constrain that we're facing. We've been ordering media in a set of dozen and imagine having two or three different media in the fridge, that'd make up to 3 X 12 bottles!

Again, thanks everyone for the input. It's final I guess, to say that if I don't paying attention to the pH, it'll will lead to paying higher price for dead cells, right.

Cheers and have a nice coming weekend

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