Stupid mistake when preparing medium - (Nov/12/2007 )
QUOTE (Travis @ Nov 12 2007, 08:42 PM)
Hi everyone,
Last week I was filtering RPMI 1640 medium for I am almost out of medium. When I was stirring the medium and calculating the pH, I forgot to add in sodium bicarbonate before measuring pH. Now I have all my medium filtered and in bottle. But the color of the medium is not as red as it should be, and that's how I realize my mistake. May I ask whats the role of sodium bicarbonate in RPMI? also, can I pour all the medium out and redo the sodium bicarbonate, pH measure, and filter steps to fix this situation? Thanks alot!!!
Travis
Last week I was filtering RPMI 1640 medium for I am almost out of medium. When I was stirring the medium and calculating the pH, I forgot to add in sodium bicarbonate before measuring pH. Now I have all my medium filtered and in bottle. But the color of the medium is not as red as it should be, and that's how I realize my mistake. May I ask whats the role of sodium bicarbonate in RPMI? also, can I pour all the medium out and redo the sodium bicarbonate, pH measure, and filter steps to fix this situation? Thanks alot!!!
Travis
RPMI has different formulas that seem to always be changing based on what is needed. Some of the powders are the only ones without certain products. I personally hate using media with phenol red in it when I am culturing mnc because I like to see the cells without the color present.
Bicarbonate is used to buffer the carbonic acid (H2C03) that forms when C02 is present in the media during culturing. The amount of (C02) is proportional to the amount of bicarbonate (HC03) used.
-dave2-
Hi Rhombus,
I don't know about prices elsewhere, but we save HEAPS by using the powder instead of buying media ready to use.
The MEM we use is $38 for powder to make 10L, and we make up usually 10L at once
(sometimes only 5, if the lab isn't flat out with tissue culture at that time).
To buy 10L ready made is $478.
The vacu-cap filters we use are about $17 each, and I can filter the whole 10L with one.
The only other thing I need to add is sodium bicarb (which, for 10L is about $2 worth).
Takes me about 1 hour to make (if its a slow day 
) and cost to the lab for my labour is $23 per hour.
So when you take all of that into account, it costs us $80 to make 10L of media,
which saves the lab just under $400.
Well worth the effort I would say
-lauralee-
QUOTE (lauralee @ Nov 14 2007, 04:02 PM)
Hi Rhombus,
I don't know about prices elsewhere, but we save HEAPS by using the powder instead of buying media ready to use.
The MEM we use is $38 for powder to make 10L, and we make up usually 10L at once
(sometimes only 5, if the lab isn't flat out with tissue culture at that time).
To buy 10L ready made is $478.
The vacu-cap filters we use are about $17 each, and I can filter the whole 10L with one.
The only other thing I need to add is sodium bicarb (which, for 10L is about $2 worth).
Takes me about 1 hour to make (if its a slow day ) and cost to the lab for my labour is $23 per hour.
So when you take all of that into account, it costs us $80 to make 10L of media,
which saves the lab just under $400.
Well worth the effort I would say
I don't know about prices elsewhere, but we save HEAPS by using the powder instead of buying media ready to use.
The MEM we use is $38 for powder to make 10L, and we make up usually 10L at once
(sometimes only 5, if the lab isn't flat out with tissue culture at that time).
To buy 10L ready made is $478.
The vacu-cap filters we use are about $17 each, and I can filter the whole 10L with one.
The only other thing I need to add is sodium bicarb (which, for 10L is about $2 worth).
Takes me about 1 hour to make (if its a slow day
) and cost to the lab for my labour is $23 per hour.So when you take all of that into account, it costs us $80 to make 10L of media,
which saves the lab just under $400.
Well worth the effort I would say
You are correct Lauralee in stating that powders are cheaper then already prepared media, but it depends where you are buying it.
-dave2-
True, dave2.
To be honest, we haven't really looked at buying the media from another company- as the one we currently use works really well for our cells, and the price is really good!!
-lauralee-
QUOTE (lauralee @ Nov 14 2007, 05:02 PM)
Hi Rhombus,
I don't know about prices elsewhere, but we save HEAPS by using the powder instead of buying media ready to use.
The MEM we use is $38 for powder to make 10L, and we make up usually 10L at once
(sometimes only 5, if the lab isn't flat out with tissue culture at that time).
To buy 10L ready made is $478.
The vacu-cap filters we use are about $17 each, and I can filter the whole 10L with one.
The only other thing I need to add is sodium bicarb (which, for 10L is about $2 worth).
Takes me about 1 hour to make (if its a slow day ) and cost to the lab for my labour is $23 per hour.
So when you take all of that into account, it costs us $80 to make 10L of media,
which saves the lab just under $400.
Well worth the effort I would say
I don't know about prices elsewhere, but we save HEAPS by using the powder instead of buying media ready to use.
The MEM we use is $38 for powder to make 10L, and we make up usually 10L at once
(sometimes only 5, if the lab isn't flat out with tissue culture at that time).
To buy 10L ready made is $478.
The vacu-cap filters we use are about $17 each, and I can filter the whole 10L with one.
The only other thing I need to add is sodium bicarb (which, for 10L is about $2 worth).
Takes me about 1 hour to make (if its a slow day
) and cost to the lab for my labour is $23 per hour.So when you take all of that into account, it costs us $80 to make 10L of media,
which saves the lab just under $400.
Well worth the effort I would say
Dear Lauralee,
Your paying far too much for your media. In the UK in our Institute we pay £2.50 for 1x500ml of RPMI/DMEM/MEM...i.e. $5.74 Australian. I make that $114.8 for 20 bottles (10L)....your getting ripped off if you are paying $478.
Also the quality control from the made-up media is going to be alot higher than many people making up powdered media.....just another variable to introduce into the mix.
AND before you say anything, our Institute is in a University environment and our money comes from grant awards, so we our always trying to get value for money.
Kindest regards
Rhombus
-Rhombus-
If you buy a lot of medium, you can get interesting prices.
Often several labs associate together to create a kind of shop that will buy these common furnitures for them, as you will get a better price if one buy a lot than if several labs buy a few.
-Missele-
I did not prepare medium by myself. But it seen to be easier to make 1L of 10X medium and then dilute it with autoclaved water. Am I right?
-WHR-
QUOTE (WHR @ Nov 15 2007, 08:57 AM)
I did not prepare medium by myself. But it seen to be easier to make 1L of 10X medium and then dilute it with autoclaved water. Am I right?
that depends on the solubility of the components. you may not be able to solubilize some of the components to 10x the working concentration.
-mdfenko-
QUOTE (WHR @ Nov 15 2007, 06:57 AM)
I did not prepare medium by myself. But it seen to be easier to make 1L of 10X medium and then dilute it with autoclaved water. Am I right?
Question: What level of pyrogen in the water are you adding to the 10X ? Do you test for Endotoxin by Limulus testing ?
I ask everyone on this thread if they test for endotoxin after making up their powdered medias ? All pre-made medias are endotoxin tested and are therefore thoroughly quality controlled. You can buy Distilled water from Invitrogen which is pre-tested , but form WHR post, it looks like he uses normal distilled water that he later autoclaves ? Are duran bottles used, if so are they thoroughly oven baked to reduce endotoxin contamination ? As I said before in my earlier post you are liable to increase variabilty in experimentation by doing procedures like this.
Thoughts please
Rhombus
-Rhombus-
QUOTE (lauralee @ Nov 14 2007, 05:14 PM)
True, dave2.
To be honest, we haven't really looked at buying the media from another company- as the one we currently use works really well for our cells, and the price is really good!!
To be honest, we haven't really looked at buying the media from another company- as the one we currently use works really well for our cells, and the price is really good!!
Dear Lauralee,
I am enjoying this online debate on what I thought nobody does anymore. Again in the academic Institute I work in NOBODY (approx 180 scientists/technicians) makes up powdered media. In the 1970's when making Polio vaccine in Industry we made up powdered media, but we were using between 150 and 200L of media and pre-made media was very expensive. But they were in the dark ages when we used Glass for everything.....TC flasks, pipettes, cryogenic storage of cells etc. In those days we cultured cells on the bench with just bunsen burners to sterilise the glass.
It's easy now with pre-made media, 0.2uM filtered tops for TC flasks, disposable pipettes, Class II cabinets.
Why go back to the dark ages.
-Rhombus-