does RPMI need to be stored away from light?

[kanditry]

Hi members
does anybody have information about why RPMI and cell medium in
general are stored in the dark away from room-lights? Our cell culture
distributor is unable to offer a credible answer...
In our busy lab we have a well light walk-in cold-room that is always
well illuminated where all our refrigerated cell culture stuff
(including medium) is stored. Will this lead to problems in the
future?

[Najfeld, Vesna]

No

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[Dunn, Betty]

According to Culture of Animal Cells, 4th Ed, 2000, by R. Ian Freshney some forms of fluorescent lighting will cause riboflavin and tryptophan to deteriorate into toxic byproducts- Freshney quotes Wang, 1976. So, the recommendation is: incandescent lighting should be used in cold rooms where media are stored, in incubators where cells are cultured and lights should be kept on a minimum of time. To guard against such exposures after manufacture, during transit and storage some media are supplied in colored light resistant plastics covers. Total exposure should not exceed a few hours. A dark refrigerator or freezer is best - so say the experts! Maybe you could construct some easily accessible light blocking storage inside your lighted walk-in refrigerator.

Betty

-----Original Message-----
From: cytogenetics-methods...@googlegroups.com [mailto:cytogenetics-methods...@googlegroups.com] On Behalf Of kanditry
Sent: Sunday, February 13, 2011 11:57 PM
To: Cytogenetics methods and trouble-shooting
Subject: does RPMI need to be stored away from light?

--

[K H Ramesh]

Hi:
I think it is because of L-glutamine which polarizes when exposed to
long term light especially UV. Even if expposed it should not bring in
big problems.
Hope this helps.
Ramesh

>>> kand...@gmail.com 02/14/11 12:56 AM >>>

[Saqib Mahmood]

Hi All

 

Further to this question, I would like to ask whether it is possible to freeze the basal RPMI media into alequote of   5 or 10 ml, thaw on the day of setting up culture and add the ingredients to make it complete media. I am asking this becuase once the bottle of RPMI is opened and we do not have many cultures to set up, the media changes it color after sometime( I beleive pH is altered) and sometimes there is growth in medial also.

 

Thank you & Best regards

 

Saqib

--
Dr Saqib Mahmood
Department of Human Genetics & Molecular Biology
University of Health Sciences
Khayaban-e-Jamia Punjab
Lahore - Pakistan
Phone: 9231304 - 09
UAN: 111 33 33 66
Mobile: 0300-4524556

[rodriguezdosiky]

u most certainly can.
All medium can be frozen but you need to check with your medium
supplier.
You might still want to add the L-glutamine once the medium is thawed.
We prepare RPMI for freezing as follows-
Add 15% FBS + RPMI 1640 + antibiotics + L- glutamine and dispensed
into 10ml tubes which are frozen at -30C for no more than 6 months

On Feb 19, 8:03 pm, Saqib Mahmood <snn...@gmail.com> wrote:
> Hi All
>
> Further to this question, I would like to ask whether it is possible to
> freeze the basal RPMI media into alequote of   5 or 10 ml, thaw on the day
> of setting up culture and add the ingredients to make it complete media. I
> am asking this becuase once the bottle of RPMI is opened and we do not have
> many cultures to set up, the media changes it color after sometime( I
> beleive pH is altered) and sometimes there is growth in medial also.
>
> Thank you & Best regards
>
> Saqib
>

> On Tue, Feb 15, 2011 at 3:29 AM, K H Ramesh <kram...@montefiore.org> wrote:
>
>
>
>
>
> > Hi:
> > I think it is because of L-glutamine which polarizes when exposed to
> > long term light especially UV. Even if expposed it should not bring in
> > big problems.
> > Hope this helps.
> > Ramesh
>

> > >>> kandi...@gmail.com 02/14/11 12:56 AM >>>

> >  Hi members
> > does anybody have information about why RPMI and cell medium in
> > general are stored in the dark away from room-lights? Our cell culture
> > distributor is unable to offer a credible answer...
> > In our busy lab we have a well light walk-in cold-room that is always
> > well illuminated where all our refrigerated cell culture stuff
> > (including medium) is stored. Will this lead to problems in the
> > future?
>
> > --
> > You received this message because you are subscribed to the Google
> > Groups "Cytogenetics methods and trouble-shooting" group.
> > To post to this group, send an email to
> > cytogenetics-methods...@googlegroups.com.
> > To unsubscribe from this group, send email to
> > cytogenetics-methods-and-t...@googlegroups.com.
> > For more options, visit this group at
>

> >http://groups.google.com/group/cytogenetics-methods-and-trouble-shoot...
> > .

>
> > --
> > You received this message because you are subscribed to the Google Groups
> > "Cytogenetics methods and trouble-shooting" group.
> > To post to this group, send an email to
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> > For more options, visit this group at

> >http://groups.google.com/group/cytogenetics-methods-and-trouble-shoot...
> > .

>
> --
> Dr Saqib Mahmood
> Department of Human Genetics & Molecular Biology
> University of Health Sciences
> Khayaban-e-Jamia Punjab
> Lahore - Pakistan
> Phone: 9231304 - 09
> UAN: 111 33 33 66

> Mobile: 0300-4524556- Hide quoted text -
>
> - Show quoted text -

[Crawford, Lorna]

Hi,

We are having issues with our prenatal preparations in particular at the
moment. They seem to be covered in debris which we are assuming is the
Leishmans stain precipitating back into its original components and
sticking to our preparations. I've included an image for information.

Is anyone else having issues with this? Does anyone have any suggestions
on how to reduce this? We have tried filtering the stain before use, we
always skim the top to reduce the precipitation but nothing is working.
We are now being forced to change the stain every 20 minutes which is
time consuming and expensive, but we have yet to analyse the results of
this.

Any suggestions will be gratefully received.

Lorna.

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[Davis, Sue D.]

We also have this problem occasionally and we have come to the
conclusion that the powdered stain is too old so we open a new bottle
and the problem goes away. We also try to keep the stain covered to
reduce precipitation and keep the entire coplin jar covered (we use a
small cardboard box) to keep the stain from too much light. It seems to
work most of the time but we still run into the issue now and then. I'd
be curious to know if anyone experiences this issue with Giemsa? We
have been using Leishmans in this lab since I started working here (15
years). I have never heard of other labs using it. Giemsa seems to be
the stain of choice for the majority of labs.

-----Original Message-----
From: cytogenetics-methods...@googlegroups.com
[mailto:cytogenetics-methods...@googlegroups.com] On

Hi,

Lorna.

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[trblue]

Leishman's stain will form a precipitate when added to pH 6.8 Gurr
buffer. Therefore, the two should not be mixed until just prior to
flooding the slide. Ensure they are mixed well. Sunlight and air will
over a period of time lead to a percipitate as well. Use a smaller
coplin jar to reduce waste. Leishman's is prone to peripitation when
it gets closer to its expiry date...try using a fresher batch lot.


Our solid staining procedure:


a)Stain slides 3 to 5 minutes in working stain.

b)Rinse well in buffer. If stain is too intense, wash longer in
buffer. If it is too weak, restain the slides.

c)Blot dry with bibulous paper. Air dry completely.

d)Rinse in xylene.

e)Mount in neutral mounting medium.

Reference:

Gustashaw, KM. Chromosome Stains. In The ACT Cytogenetics Laboratory
Manual, Second Edition, edited by M. J. Barch. The Association of
Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.


On Feb 23, 4:38 am, "Crawford, Lorna" <Lorna.Crawf...@ggc.scot.nhs.uk>
wrote:

> Hi,
>
> We are having issues with our prenatal preparations in particular at the
> moment. They seem to be covered in debris which we are assuming is the
> Leishmans stain precipitating back into its original components and
> sticking to our preparations. I've included an image for information.
>
> Is anyone else having issues with this? Does anyone have any suggestions
> on how to reduce this? We have tried filtering the stain before use, we
> always skim the top to reduce the precipitation but nothing is working.
> We are now being forced to change the stain every 20 minutes which is
> time consuming and expensive, but we have yet to analyse the results of
> this.
>
> Any suggestions will be gratefully received.
>
> Lorna.
>

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> All messages passing through this gateway are checked for viruses, but
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>

>  debris.tif
> 2214KViewDownload

[Crawford, Lorna]

Thank you for your replies. We are experiencing this issue with newly opened/ ordered powder so it does not seem to be related to the age of the Leishmans. I should have mentioned that we are also using a banding machine which includes a large pot of stain in the process so mixing the buffer and Leishmans just before staining in a small coplin is not possible.

We have not had this issue from the point of moving onto the machine so I'm confused as to why suddently the Leishmans is sitting too long, especially since we change it more now than we did at the start.

We have ordered Giemsa to try, I'll keep you posted with the results.

Lorna.

-----Original Message-----
From: cytogenetics-methods...@googlegroups.com [mailto:cytogenetics-methods...@googlegroups.com] On Behalf Of trblue
Sent: 23 February 2011 00:37
To: Cytogenetics methods and trouble-shooting

Subject: Re: Leishmans stain

Our solid staining procedure:

d)Rinse in xylene.

Reference:

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[ramakrishnan sasi]

 

Ramakrishnan Sasi, Ph.D., FACMG
916 Cleveland Avenue # 7
Marquette, MI 49855
 

---

From: "Crawford, Lorna" <Lorna.C...@ggc.scot.nhs.uk>
To: cytogenetics-methods...@googlegroups.com
Sent: Wed, February 23, 2011 4:50:45 AM
Subject: RE: Leishmans stain

To unsubscribe from this group, send email to cytogenetics-methods-and-trouble-shooting+unsub...@googlegroups.com.

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