lyophilizing cultures

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Sun Jul 8 05:35:04 EST 2001


In article <006e01c10714$84f566a0$8c01a8c0 at tn.nic.in>, the eminent R.
Jayakumar at School of Biotechnology wrote
>Somebody told me that I should add 2% milk powder to the culture and then do
>the lyophilization.  is that true? 

People use milk powder, serum etc.

> Anyway, I am facing problems.  I first
>grow the culture in 2 ml LB or NB or .... overnight, and then add sterile
>10X stock of milk powder solution to a final conc. of 2%. I take 0.5 ml of
>this in the virtis long stemmed sterile glass ampule, close it with cotton
>and freeze it quickly in -70C.  Then I fix it to one of the vacuum outlets
>on the lyophilizer and start the drying process. I faced several problems.
>  1.  The culture somehow thaws and starts bubbling badly.. virtually all
>the culture in some tubes gets sucked upwards and absorbs into the cotton
>during drying due to the vacuum. I am losing the cultures substantially.
>How do I prevent this?

I believe most small culture collections lyophilise whilst centrifuging
the culture, at low speed, for initial drying then put the ampoule onto
a secondary vacuum apparatus for sealing. That is what I have here,
(unused!) as we could never get hold of the Edwards machine that flamed
and pulled out ampoules. Doing it manually was not on at the time we
were looking at doing this. 

I'm still looking for one of these if anyone has one unused taking up
cupboard space. 

>   2. Can I add anything else other than milk powder to the solution?  I
>need some real quick replies for this... since i need to do and finish it by
>tomorrow.

Have you looked at the ATCC web site. They may have info as to what they
use.

Duncan
-- 
Homogeneous Fluorescent Reporting Systems for Real-Time Quantitative PCR: 

Optimisation, Probe Technology & Future Systems

4-5 September 2001
King Alfred's College, Winchester, UK 

http://www.dera.gov.uk/html/news/events/real_time_quantitative_pcr.htm




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