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.
