4 replies to this topic

[bokashi farmer]

I cant figure out why my media turn alkaline, instead of acidic.

Im culturing MCF-7 (ATTC,USA) in EMEM (Mediatech, USA) supplimented with 5% FBS (PAA,USA) & 1% pen-strep (PAA,USA) incubated in 37degC, 5% CO2. After 24h, they turn pinkish and cloudy. The cells start to lyse, debrise formed and if I leave it for 48hr, all cell lysed, media get more alkaline. When I used 10% FBS (Im supposed to use this percentage in my research), media gets cloudy, 50% cells floated. If I try 15%, cells died in 24h. I checked old media's pH daily. pH climbed from 7.4 to 7.8 to 8.2 the following days.

Im using T75 flask, had to loosened the caps. If not cells near the cap will confluent but cells at the back of the flasks lysed. Suspected that the cells are not getting enough CO2.

I only get confluent cells, hassles-free if I gave the cells 5% FBS, seed at higher seeding density, loosen the cap, dont spray ethanol before keeping the flasks in the incubator & change media daily. No floating cells, no debrise, 80% confluence at 2days. But why? I cant keep doing this, I'll be using a lot of media quickly. Finding the balance is time consuming, I need to keep moving. I have similar problem with MDA-MB-231.

I really need help, guys.

Edited by bokashi farmer, 04 September 2010 - 09:21 PM.

[Curtis]

im not sure if I understand your problem exactly but there are a couple of reasons why media turns alkaline:

- if there is not enough CO2 in incubator. Also usually when you take our your flasks out to work under laminarflow they turn alkaline after some time. but if you put them back in incubator they turn neutral

- when cells die and there is no cell to consume the nutrition the media remains same color as your stock media. When cells consume nutrition and are healthy the media becomes acidic and you need to add fresh media.


my suggestions:
- if your T75 flask has filter cap then do not leave your cap loose.
- put your flask at the end of incubator, not near the door
- put it in higher level where
- Use DMEM for mcf-7

[bob1]

Bacterial contamination is the most likely problem... many bacteria will turn the medium alkaline.

[leelee]

I agree with bob1. You said your media was cloudy after 24 hours- that screams contamination to me.

Edited by leelee, 05 September 2010 - 06:28 PM.

[briguy7]

Cloudy = contamination