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Difco Marine broth 2216 precipitate

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#1 GaryD

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Posted 25 May 2014 - 06:39 AM

Hi,

I just bought some Difco Marine broth (2216), and tried preparing it for the very first time. I followed the manufacturer's instructions (37.4g dissolved in 1 l, mix thoroughly, heat, boil for 1 min to completely dissolve the powder). Unfortunately, however, I found that the mixture forms a pretty thick yellow precipitate, which isn't removed by the boiling; it's as turbid as a bacterial culture would be! I tried various things to get rid of this precipitate: different water (including milli-Q), checking the pH and changing to 7.6, heating slow and fast, boiling for over a minute, all to no avail. I even tried autoclaving the mixture as was, and the precipitate obviously remained.
Does anyone have any ideas? Or has anyone used this mixture and had the same problems? I've read various things that seem to suggest some precipitate could be normal, but this seems really a lot to me. As I say I've followed the instructions, so I'm not sure what I could be doing wrong, and I'm now wondering if this is just a result of what is contained in the media. Anyone with experience, please let me know!
Here is a list of the media composition:

 

Difco™ Marine Broth 2216
Approximate Formula* Per Liter
Peptone ......................................................................5.0 g
Yeast Extract ...............................................................1.0 g
Ferric Citrate ...............................................................0.1 g
Sodium Chloride .......................................................19.45 g
Magnesium Chloride ...................................................5.9 g
Magnesium Sulfate .....................................................3.24 g
Calcium Chloride ........................................................1.8 g
Potassium Chloride .....................................................0.55 g
Sodium Bicarbonate ....................................................0.16 g
Potassium Bromide ......................................................0.08 g
Strontium Chloride ....................................................34.0 mg
Boric Acid .................................................................22.0 mg
Sodium Silicate ............................................................4.0 mg
Sodium Fluoride ..........................................................2.4 mg
Ammonium Nitrate .....................................................1.6 mg

Disodium Phosphate ...................................................8.0 mg   



#2 phage434

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Posted 25 May 2014 - 06:45 AM

This is normal for marine broth. I suppose you could sterile filter it, but the cloudiness is normal.



#3 GaryD

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Posted 25 May 2014 - 06:54 AM

Thanks. But also, crikey, really?! I just can't believe you can grow anything in it, it's so cloudy. When I mix it up at all, it's completely opaque. Can anything be done to reduce the cloudiness? I hate the thought of filtering it, as I assume I'd then be removing stuff that the bacteria might actually need.



#4 phage434

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Posted 25 May 2014 - 07:01 AM

Are you using the Difco pre-mix, or making this yourself? The likely suspects for precipitation are anything with iron in it, and the magesium or calcium phosphate. For plates, the opacity matters little, since the agar is also not transparent. The precipitate falls out of solution when not shaken, and I've not noticed a problem in culturing things with the clear supernatent.



#5 GaryD

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Posted 25 May 2014 - 07:13 AM

I'm using the Difco pre-mix. I guess my main worry is that getting reliable OD measurements will be impossible using the media as it is, even if I let it settle before each measurement, because the precipitate is just so thick (as I say almost as bad as a stationary-phase culture!). I'm also worried that there's so much precipitate it could potentially provide a substrate for attachment, which could further complicate growth. I've seen reports of a 'slight precipitate', but I'm surprised anyone can use this as it is without filtering or some other measures, but no papers seem to report this from what I've seen. I'm thinking about preparing it myself from scratch, and trying to make separate mixtures and avoid precipitation by mixing them after autoclaving, but I have a feeling this might prove somewhat complicated given the complexity of the mixture!

Once again, many thanks. 



#6 phage434

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Posted 25 May 2014 - 07:20 AM

It sounds as if there is way more precipitate than I normally see. Is your scale accurate? Tared? To be precise, I see a thin (< 1 mm) layer barely covering the bottom of a 1 liter media bottle after things have settled out.



#7 GaryD

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Posted 25 May 2014 - 07:31 AM

As far as I'm aware the scales are accurate, but certainly I'll try different scales as an alternative measure. I think that once settled, I'm getting a similar amount of precipitate to you, which still seems like an alarming amount when mixed, especially as the manufacturer's instructions seem to be suggesting that boiling should dissolve all the powder.


Edited by GaryD, 25 May 2014 - 07:50 AM.


#8 El Crazy Xabi

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Posted 25 May 2014 - 05:54 PM

From the Difco culture media manual

 

Difco™ Marine Broth 2216
Dehydrated Appearance: Light beige with a few dark particles, free flowing, homogeneous.
Solution: 3.74% solution, soluble in purified water upon boiling. Solution is light amber, slightly opalescent with precipitate.
Prepared Appearance: Light amber, slightly opalescent with a precipitate.
Reaction of 3.74%
Solution at 25°C: pH 7.6 ± 0.2

 

So, that's normal.

 

If you don't want precipitate you would need to add any divalent cation, heavy metal, etc, separately autoclaving them individually or filter sterilizing. With the commercial mix you will get a precipitate of Mg, Ca, Sr(less likely) and Fe phosphates, as well as their sulfates. You will not be able to change it unless you modify the preparation method as suggested by phage, i.e. filtering







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