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Bacteria pellet has almost-black region


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#1 ah6tyfour

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Posted 26 April 2009 - 12:41 AM

Hi everyone,

I'm planning on doing some MaxiPreps so I grew up a bacteria colony in 250mL of LB with Amp at 37-degrees for 16 hours. I could not immediately spin down the liquid cultures, so I stuck them in the fridge for 12 hours. I then spun it down at 4000rpm for 20 minutes in a refrigerated centrifuge and decanted the media.

As I go to freeze down my pellet, I notice that the pellet isn't the same color throughout. Most of it is the usual tan color, but there are areas that are dark grey or almost black. Is this normal?

I usually just do minipreps, but am doing maxipreps this time because the plasmid will be used for transfection. I don't recall ever getting bacteria pellets that looked anything other than nicely tan throughout.

I'm just wondering if maybe they grew past the exponential stage or are somehow contaminated...hopefully it's not the case.

At first I just thought it was some black Sharpie mark on the outside so as I look at the pellet from the inside, I can see the black mark show through. But there is no mark on the outside at all!

To make things even more strange, only three out of four of my pellets have this strange dark color. The fourth one is nicely tan.

Thanks!

#2 HomeBrew

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Posted 26 April 2009 - 07:47 AM

What organism is it? What media was it grown in?

#3 mastermi

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Posted 26 April 2009 - 12:21 PM

I often have dark smear in my pellets when I picked colonies with toothpicks into liquid media. In that case the darker colour is due to the toothpicks.
I don't know what this colour may come from in your pellets, but I wouldn't store a liquid culture in the fridge before extracting plasmids, and definitly not for 12 hours!
If you want to make sure that you get high ammounts of clean plasmid, always centrifuge your cultures as soon as possible...

#4 ah6tyfour

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Posted 26 April 2009 - 04:57 PM

thanks for the rely mastermi. I picked my colonies with pipette tips so there are no chemicals from the toothpicks in my media (although I used to use toothpicks in a former lab and liked it much more than using tips). But the type of dark smear you are describing sounds exactly like what I have.

I regrew 250mL of LB with some of my pellet from the previous time and this time I spun it down immediately. The same dark smear is still there. So I guess they should all be okay....

How do you know your dark smear is from the toothpicks?

Thanks!

#5 HomeBrew

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Posted 26 April 2009 - 05:30 PM

The reason I asked what bug it was and what media is because we see that all the time with Bacteroides cultures, but the media is supplemented with iron, which we suspect is the cause. In our cultures, it's not a sign of contamination. I used to use toothpicks as well, and never noticed this phenotype with, say, E. coli cultures. I'm using pipette tips now 'cause everyone else in the lab does, but I still prefer toothpicks.

#6 ah6tyfour

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Posted 26 April 2009 - 09:51 PM

Yea, just plain ol' E. Coli (XL10-gold...I'm not particularly fond of this strain).

I don't know why it has become popular to pick colonies with pipette tips. I prefer toothpicks too. Toothpicks offer better grip and you can angle them just right to nicely pick up a colony. Pipette tips are sort of a pain to hold and I hate that some LB moves into the tip through capillary action. Some of my coworkers actually hold the pipette tip by attaching it to the micropipettor and holding the pipettor as if they were going to pipet liquid. How they are able to nicely remove a colony that way is beyond me, but they do it.

I might just cave in one day and go to the store and buy myself some toothpicks....

#7 mastermi

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Posted 27 April 2009 - 09:50 AM

thanks for the rely mastermi. I picked my colonies with pipette tips so there are no chemicals from the toothpicks in my media (although I used to use toothpicks in a former lab and liked it much more than using tips). But the type of dark smear you are describing sounds exactly like what I have.

I regrew 250mL of LB with some of my pellet from the previous time and this time I spun it down immediately. The same dark smear is still there. So I guess they should all be okay....

How do you know your dark smear is from the toothpicks?

Thanks!


I don't know it, it was just a guess because I always see it when using toothpicks, and never saw it when using pipette tips (for E. coli).
But if you still see it it must be due to anything else, I have no idea what...

#8 hanming86

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Posted 29 April 2009 - 07:14 AM

I recalled seeing it when i was doing my maxi prep too. But i don't htink it inteferes with as far as i am concerned restriction digest or ligation. Probably caused by the LB ( different batch got sligh different composition ) Iron precipates sometimes.
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#9 ah6tyfour

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Posted 01 May 2009 - 11:19 PM

Reporting back....there doesn't seem to be any ill effects of whatever the dark streak was.

Ironically the one sample that didn't have that streak didn't work. But it was my fault. I apparently had too many cells so cell lysis didn't go well (I could see it wasn't very viscous and Qaigen's LyseBlue had a weird baby blue color instead of the normal intense blue). Stupidly I chose to continue on as is instead of having the insight to add another volume of lysis buffer.

To make a long story short....cells didn't lyse and neutralization buffer caused a homogenous mixture of individual cells which all settled to the bottom of the column...effectively clogging the whole thing from the get-go.

Lesson learned: more is not always better.

#10 swanny

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Posted 03 May 2009 - 07:31 PM

Yea, just plain ol' E. Coli (XL10-gold...I'm not particularly fond of this strain).

I don't know why it has become popular to pick colonies with pipette tips. I prefer toothpicks too. Toothpicks offer better grip and you can angle them just right to nicely pick up a colony. Pipette tips are sort of a pain to hold and I hate that some LB moves into the tip through capillary action. Some of my coworkers actually hold the pipette tip by attaching it to the micropipettor and holding the pipettor as if they were going to pipet liquid. How they are able to nicely remove a colony that way is beyond me, but they do it.

I might just cave in one day and go to the store and buy myself some toothpicks....

Pipette tips vs toothpicks is often a matter of practice...
Don't forget, when you get the toothpicks, go for the cheapest ones... less likely to have antibiotics impregnation!
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#11 madrius1

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Posted 04 May 2009 - 10:41 AM

I've seen this kind of "black area" in my cells a lot of times. I wonder what it was, but it surely never alterated my results. I use DH5alpha cells, so good'ol E Coli also!




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