Culture without CO2 incubatore

[Mol cyto]

 

 

Dear FriendsI would appreciate if someone comment on how to culture cells without CO2 incubatore. Is it advisable to do blood cultures in normal incubator for karyotyping which is just for student's demo class.... because I recently joined in this lab where they have no CO2 incubator and did cell culture wok......

Thank you

 

[Winnie Lam]

It is possible to do blood cell culture in ordinary incubator (without CO2), as it is closed culture. Winnie --- 2010年2月22日 星期一，Mol cyto <molc...@gmail.com> 寫道﹕ 寄件人: Mol cyto <molc...@gmail.com> 主題: Culture without CO2 incubatore 收件人: cytogenetics-methods...@googlegroups.com 日期: 2010年2月22日,星期一,下午3:25

Dear FriendsI would appreciate if someone comment on how to culture cells without CO2 incubatore. Is it advisable to do blood cultures in normal incubator for karyotyping which is just for student's demo class.... because I recently joined in this lab where they have no CO2 incubator and did cell culture wok...... Thank you

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[Dunn, Betty]

Blood cell cultures may be grown in a closed container (tube or flask) so there is no exchange of air from inside the container with air outside the container for 2-4 days (never open the culture container until time to harvest). Cultures must be maintained at 37oC, which can be done in a waterbath, for instance, or a non-CO2 incubator.   Media must have at least a buffering system, bicarbonate is typical, so that CO2 generated by metabolizing cells escapes into the enclosed air and interacts with the media buffering system to maintain an adequate pH in the media.  Other buffering systems are much more expensive.  Our standard media all have bicarbonate buffering systems.       

 

For long term cultures in closed containers, cultures must have the airspace above the media replaced with CO2 as the cellular generated CO2 is inadequate for long term culture.  So, long term cultures must be “gassed” from pressurized CO2 tanks periodically to maintain adequate conditions for growth.   This method is very time consuming and not as proficient or consistent as growing long term cultures in open culture systems in a CO2 incubator.   

 

Betty Dunn, MS, CLSp(CG)

Associate Professor

Director, Cytogenetics Program

UTHSCSA

Dept. of Clinical Laboratory Sciences

7703 Floyd Curl Dr.

San Antonio, TX 78229-3900

210-567-8865

210-567-8875 (FAX)

--

[vantuinen]

Blood or marrow cultures can be done without CO2 incubators. Normally
they produce enough of their own CO2 metabolically. Eeva Therman (Mrs
Patau) used to culture blood in 15 ml tubes with the cap initially
closed. Each day the color of the medium was checked, if it was too
yellow the cap was loosened a bit, if red it was left tight. Longer
term cultures can also be performed with no CO2 source. A trick I
have used for years successfully is as follows: Hold your breath for
as long as possible, exhale ½ way to clear your your trachea of dead
air, then exhale the CO2-rich air through a sterile cotton plugged
pipet into the flask. The CO2 concentration will be correct, the
oxygen will be lower (remember, lower than ambient oxygen tension is
preferred). You have just achieved what a 3-gas incubator does. IF
you think this is bogus try it on a flask with fresh medium and note
the color change to yellow or peach after a couple of minutes.
Pete vanTuinen

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