[Microbiology] Re: media preparations

John Gentile via microbio%40net.bio.net (by yjgent from nospamcox.net)
Tue Jan 8 21:00:05 EST 2008


On 2008-01-08 11:35:30 -0500, deepgolwala from gmail.com said:

> i am new to the field ,
> i would like to ask while making LB agar(tryptone-10 gms,yeast
> extract-5 gms,NaCl-10 gms-all weight for 1000ml preparations)when
> should i add agar during preparations or after autoclaving.

Making media is one of the things I truely enjoyed in my early days in 
the micro lab. With experience you will pick up a lot of good tips on 
making quality media - usually better than any factory made! A few 
points I can pass along,
- agar melts around 96 C, and solidifies around 45-50C. so make sure 
you add the agar to your mix and boil it - but watch for over boiling! 
Then autoclave it in a round flask to avoid boilover (Erlenmeyer Flasks 
and "Fleakers" are known for this). Don't seal the flask tight, but use 
a gauze plug in the mouth to allow steam and pressure to equalize. Cool 
the solution in a 56C waterbath and decant when cooled. I used to use a 
spring loaded refilable syringe to make plates and tubes, but hand 
pouring is pretty good too. If you get bubbles in the media you need to 
use a bunsen burner to flame the bubbles off before the media 
solidifies.
- some media contains bile salts that precipitate out with heat. These 
I heated only to melt the agar and poured immediately. XLD is an 
example, and did not need autoclaving.
- some media have added ingredients that cannot be boiled or 
autoclaved. These need to be filter sterilized and then added after the 
autoclaved media has cooled and then can be decanted.

Get a good media book that contains recipies and also preparation tips. 
I always used Difco and BD's book, but I don't know what is out there 
now!

-- 
John Gentile MS, M(ASCP)
Laboratory Information Mgr.
VA Medical Center
Providence, RI 
yjgent from cox.net



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