Cloudy NZY + Broth maybe contaminated?? - (Jul/28/2011 )

Hello all. I have made NZY+ broth for my XL10-Gold Cells twice now and both times it appears to be very very easily contaminated. Here if my recipe:

10g NZ Amine (casein hydrolysate)
5 g yeast extract
5 g NaCL

add h20 up to 1 L, adjust pH to 7.5 with NaOH, and then autoclave.

Prior to use, I add filter sterilized in a hood:

12.5ml 1M MgCl2
12.5 ml of 1M MgSO4
10 ml of 2M glucose

I used it one time by opening quickly and taking what I needed and immediately resealing (all done in open air). A few days later I noticed that it was a little cloudy so I remade it exactly as described above, but this time when I aliquoted some I took it into the hood, sprayed everything with EtOH, had brand new gloves sprayed with EtOH and only had bottle open for 5 seconds. Again, about a day or two later I noticed it is cloudy again! Is this media very very very easy to contaminate, or is it some ingredient (maybe the glucose) that is making the liquid cloudy?? It's driving me a little crazy because I obviously cannot remake this every time I want to transform! Tonight, I will aliquot 5 mL into one falcon tube and shake 220RPM/37deg/overnight and a separate 5ml into another falcon tube which I will let sit on my lab bench. Tomorrow I will check to see if the one that I shook is more cloudy, which I would assume means that my stock is indeed contaminated?? Any help is appreciated, as always!

Rooster

Did you filter sterilise the glucose and Mg solutions? Perhaps one of them is contaminated at a sub-visible level.

Yes I did filter sterilize them both times...does your nzy not go cloudy?

I have never specifically used NZ amine, but the similar tryptone produces a transparent solution. I was assuming that you are making something that is equivalent to SOC broth, which is also clear. Usually when making these things the Mg and glucose are added to aliquots of the medium, when needed.

I just have my technician made partial NZY+ and autoclaved (without glucose and MgSO4 and MgCl2 as suggested by QuickChange manual) and it looks like contaminated. She said it looked cloudy before she put it in the autoclave, she made it day before. But bacteria would look different after autoclaving I think (the autoclave tape is black). She was preparing 5x LB at the same time, but that is clear as usual.

Could that cloudiness be from the NZ amine? Would it help to filter it before autoclaving? I'm bit affraid to use this one, it is for the mutagenesis kit.