Pre-reduced media preparation

Robert Randolph randolph at
Fri Aug 1 08:10:42 EST 1997

Randolph Biomedical has manufactured PRAS (pre-reduced anaerobically
sterilized) media for over 30 years. It is as difficult to insure an
anaerobic media condition today as was at the start.  The key to
success is that all phases of preparation, dispensing and
sterilization MUST be done under anaerobic conditions.

#1  The medium flask must have a cannula bubbling oxygen-free CO2
until the media is reduced then oxygen-free nitrogen in the media and
head space at all times.  The media does not have to boil, but must
simmer for 20 - 40 minutes.  Always add about 10% more DI water to
compensate for water loss during reducing. 

#2  Resazurin is blue when fully oxidized, pink when partially
       oxidized and colorless when pH 6-8.

#3  Once the medium is reduced (colorless) switch from CO2 to N2.
     Place flask in cool water or ice to cool to room temp. If you
dispense hot, as the medium cools it will absorb available
gas...oxygen if present.

#4 Add cysteine, 0.5 gm per liter.  This will lower pH, you must
adjust pH to 7.1 +/1 0.2 (range for most media)  with HOT (reduced) 5N
NaOH.  CAUTION: Hot caustic will burn and burn and burn...... Remove
an aliquot and pH.  Replete until correct. Use  5N HCl to reduce pH if

#5  Tubes, bottles or flask must be purged with flowing oxygen-free
nitrogen during the filling and capping process.  All closures must be
"air tight".  If using rubber stoppers...medium must be placed in
a press rack to prevent stopper from popping when autoclaving.

$6  Do not remove from press racks until cooled to room temp.


If medium is pink and remains pink 5 minutes after dispensing, it will
not not continue dispensing. Your system has an oxygen
breach...find it and fix it.

The cysteine added to the media is not there to reduce the media, as
the heating process is suppose to do that, the cysteine is the oxygen
scavenger, to reduce any oxygen absorbed during the dispensing and
sterilizing  process.

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Thank you. R.Randolph  microbiology at


On Tue, 29 Jul 1997 14:27:15 -0700, Nisha Singh
<nisha at> wrote:

>We are seeking assistance in the preparation of pre-reduced media for the 
>cultivation of anaerobic bacteria.  We are using the preparation methods 
>of Holdemann et al., Anaerobic Laboratory Manual, 4th edn.  (1977).  We 
>have found that the indicator (resazurin) does not undergo a colour 
>change after boiling, as recorded in the manual, ie. from purple to 
>colourless in 5-10 min.  In our experiments the indicator changes from 
>purple to pink in 20 min and does not change to colourless, even after 
>prolonged boiling.  After cysteine hydrochloride addition and 
>autoclaving, the indicator in the media changed from colourless to purple 
>even when placed in an anaerobic chamber. Cooling the media under 
>nitrogen gas after boiling has also been unsuccessful. Is anyone able to 
>help us? Thank you  
>Loren Goldstone and Kimoni Rampersad
>Technikon Natal, Durban, South Africa
>E-mail: research at

 Robert Randolph    Randolph Biomedical
 21 McElroy Street   West Warwick, RI 02893
     401-826-1407   microbiology at & anaerobe.htm

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