Help freeze-drying anaerobes

Dan Beacom beacom at mbi.org
Wed Sep 20 16:15:17 EST 1995


We use the following technique:

Prep 24% sterile anearobic sucrose (boil and gas w/ N2-dispense in Bellco 
bottle-stopper and crimp)  Rezaurin (sp?) can be added as an indicator  
Autoclave.  Skim milk works well, too.

Concentrate active culture growing in bellco tubes by centrifugation (do not 
go above 4500 rpm-glass will break)  draw off 80-90% of supernatant by 
syringe).  Alot of extreme anaerobes do not reach high cell densities.

As far as apparatus, we have a glass manifold which holds 4 ampoules at one 
time.  The top of the manifold is connected to a metal tube by means of a 
black rubber hose.  Short sections of rubber hose are attatched to each of the 
4 glass tubes on the bottom of the manifold.  All five rubber hose sections 
are sealed with clamps.  The whole system is wrapped in foil and autoclaved.  
Finally it is transferred into the anearobic glove bag.

Ampoules are sterilzed empty with cotton plugs (labels may be inside) and 
placed in the anaerobic glove bag.  Get everything into the glove bag and let 
equilibrate for a couple of hours.

Using a syringe and needle, 0.1ml each of culture and sucrose are added to 
each ampoule.  The cotton plug is removed and connected to one of the 
bottom rubber tubes on the manifold.  Each tube should be uncovered only 
immediately prior to use to minimize any contamination risk.

Once the manifold is filled with four ampoules, it is removed from the glove 
bag, the ampoules are frozen (ethanol or acetone in dry ice) and hooked up to 
the lyophilizer in the following manner:
The metal rod on the manifold is connected to lyophilizer vacuum port.
The port valve is opened.  Wait for full vacuum.
Open the clamp between the metal rod and the glass manifold.  Wait for full 
vacuum.
Open the clamp to the first ampoule-wait for full vacuum.
Repeat for the other three ampoules.
Leave overnight
Heat seal ampoules per normal procedure.

We have been successful with extreme anaerobes.  It has been awhile (4-5 
years) since I have personnally performmed this procedure.  Hope it is what 
you need.  I can put you in touch with the person who performs this procedure 
most frequently if you still have problems.

Good luck,
Dan Beacom
Michigan Biotechnology Institute



In article <43ob0i$e8n at info.curtin.edu.au> Your Name <Your Email Address> 
writes:>From: Your Name <Your Email Address>
>Subject: Help freeze-drying anaerobes
>Date: 20 Sep 1995 06:06:10 GMT

>Could anyone give me some information on the successful freeze-drying (in 
>ampoules) of anaerobic organisms such as Bacteroides melaninogenicus.I 
>seem to have a hard time keeping anaerobes viable after freeze-drying. 
>Thankyou . Sue. 






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