Denny

In theory it shouldn't matter at all, so long as your sample (not standard) values are being predicted from an equation based on the standards, and within the reference range then they should remain consistent.  This is because the absorbance is relative to the concentration and is not an absolute value.

For your controls - are they giving you the same values as before?  If so, you have a problem, to which the only solution will be to get (another) batch of reagents.

However, you can check the error in the system by running a known concentration sample with your new standards and seeing what you get.

The bicarbonate concentration is dependent on the %CO₂, the atmospheric content forces the reaction away from the HCO₃^- towards the H⁺, keeping the pH buffered correctly. If there is too much bicarbonate in the medium for the %CO₂, the medium will have a higher pH (leave a small amount of medium exposed to air for a short while, with phenol red as an indicator it will go purple indicating basic conditions).  This also accounts for the colour change you see when you approach the bottom of a bottle, which then corrects itself when put in an incubator.

It is entirely possible to grow cells at the "wrong" %CO₂, the pH isn't too different, usually a difference of about 0.2-0.4 pH units between 5 and 10%

Denny thank you very much fo the useful reply!!

In our lab we use Sequencher for aligning our sequences because our university has a site license for it, but you can align the sequences without purchasing software by putting your non-coding strand in a reverse-complement calculator (like http://www.bioinform...s/rev_comp.html).  Then you can use the output in any sequence alignment site you want (BLAST or ClustalW or whatever, although they may handle converting to reverse complement themselves; I didn't check), or you can just look at your forward sequence, find a series of 8 residues or so and search for them in the second sequence.  Once you know how they match up, you just have to copy and paste the extra sequence from your reverse primer reaction onto the end of your forward sequence.

Does that make sense?  I'm sure there are good sequence alignment sites out there that would do this for you, but I'm not familiar with them.

What is it that you are quantifying with your ELISA?  Is it a therapeutic human IgG that binds to a target and your coating peptide is a portion of that target?  Just trying to understand your ELISA.

The combination of your biotinylated antibody and streptavidin-HRP in a pre-incubation/complexing step may be unrobust and will need titering for each lot used or perhaps you should consider avoiding the complex thing completely.  If each lot of biotinylated antibody has a varaible free biotin component (and the free component may increase with inappropriate storage), it will variably reduce the capacity of the streptavidin to bind the antibody.  Also, the proportion of streptavidin in your streptavidin-HRP prep and number of available biotin binding sites on each streptavidin molecule (it's usually 4, but some will be blocked by the HRP), and the extent of biotinylation of your anti-human IgG (one or multiple biotins on each molecule), all will contribute the the size of your anti-human IgG-biotin-streptavidin-HRP complex and will ultimately affect the number of HRP molecules attached (indirectly) to your human IgG. If you manage to create a complex of 20 HRPs for example, then you will get a signal 20 x higher than if you don't generate a complex at all.

Someone probably once titered the streptavidin-HRP and anti-human IgG-bioin and came to the conclusion that the pre-incubation complex route gave greater sensitivity for THOSE LOTS OF REAGENTS.  It may be beneficial to make things simple, and add the anti-human IgG biotin and streptavidin-HRP sequentially with a wash step between.  In this way, there is no possibilty of free biotin left in the biotinylated antibody blocking the streptavidin, and no unreproducible complexes of biotinylated anti-human IgG/streptavidin-HRP to deal with.

So the critical question is:  has there been a lot change in either the streptavidin-HRP or the anti-human IgG?  Has the biotinylated antibody been stored incorrectly and released some of it's biotin?

More details of your assay may allow us to give additional suggestions,

good luck!

If you are talking about the dark lumps of stuff between the cells, then it is very likely to be debris from dead cells, it is a bit big for bacteria and too irregular to be most cell types, unless they are a clustering form (e.g. staphylococci).  However, the fine stuff you posted in the last image could well be contamination.

It would be unusual to note contamination after 18 hours, that is pretty quick, but there are plenty of species that could grow that fast.  The most common contaminations are from E. coli, and a few skin bacteria, many of them are motile too, so you can often see them swimming about.

Hmmm. Ok. What about increasing the centrifugation time to 10 min?

Also, I have found that centrifuge speeds that I use for my big tubes (15 and 50ml) in the larger benchtop centrifuges pellet my cells MUCH better than equivalent rcf values in a microfuge. I don't know why this is (350xg for both).

I get around it by increasing the speed for the microfuge- but by this stage my cells are usually fixed and so I don't have to worry about viability.

Hopefully someone else has some tips for you

Dear all,

It seems like many researcher faced with this bizarre black dancing dots in cell culture, and me too!
So I've search about this issue and found one paper that published in Biologicals 2010.

Gray JS, Birmingham JM, Fenton JI.Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant Biologicals. 2010 Mar;38(2):273-7. Epub 2009 Nov 18.

This one light up my life (I just order antibiotics that they mention today and hope it works. I hope this paper can help you (and me) to deal with this UFO contaminant.

Cheers!

Mypowerberry

It will be completely fine. It is actually a good way to store DNA long term.

One advise:

make sure you have anough of "proof".
If you cant catch her in the act, make sure you have "proof" that shows something is going wrong.
Use this to in a discussion with your boss.

And one hint: you dont need to accuse her right away, just collect the "proof", ask your boss to have a chat with you to discuss some strange things (without pointing the finger at her).
You can genlty mention her name , for example: we noticed some strange things with samples A and B and with the testsresults from test Y , perhaps we can debate what went wrong? Normally your boss will then ask who did those tests ....
See what I mean..
Dont start accusing her right away yourself because it can backfire + if you cant really catch her..


Also: if she comes in late and leaves early.. its something the boss needs to check, not you.
I know its not honest, but if you start complaining about it...

Denny, on 06 November 2012 - 04:54 PM, said:

hey Denny...yup, it's a tough call but as you already said, if you keep quiet then you're probably risking the good reputation of your lab and your boss. They deserve to know the truth at the very least. I'm also thinking that you shld have just busted her in real time while she was making the mistake bec if you confront her much later, it might just degenerate into your word against hers.  As you mentioned, you already have a strained relationship and this is probably not a secret to people you work with so you might appear as someone only trying to create mischief or just aiming to discredit her for your own personal reasons.

Is there a way for her to fix the mistake (cos data from contaminated samples matter and if she knowingly provided contaminated samples to be assayed then that's a form of misconduct)  so you can avoid involving the higher-ups? But I would still try to talk with her first. I know it's a hard decision but if you ignore it or let it pass, then you are condoning her action or allowing her to think that she can get away with anything and in the end, you are just as guilty plus you wouldn't feel good about yourself either. Anyways, doing the right thing is never easy for anybody but hopefully we have the courage of our convictions.

yeah... but you don't know who took them out and in after 1-2 hours in the weekend without saying anything (I have seen similar things happening). I would inoculate in a liquid culture directly from the stock and see what happens. On the other hand, I would never trust that culture if I do not see single bacterial colony on the plate. On the other hand, if you still have the DNA, what would stop you from getting a new transformation? It might be a bit too much work, but better do it again than forever wonder whether what you are working with is the real thing and about where the problem is. Just my two obsessive compulsive-paranoid cents...

Andreea