I am freeze drying mixed bacterial cultures in 10-20% non fat skim milk. I grow my different cultures in TSB broth and extract the bacterial cells by centrifuging the broths discard supernatant and suspend pellets in skim milk. After mixing pellets with the milk for each organism I then mix the different suspensions in to one tube from which I measure 0.5ml to prefreeze. The final mixed suspension is first cooled in lab freezer before being transferred to the ultra freezer(-80-oC) where it stays overnight. The following day I switch on the refrigeration of the freeze dryer to lower the temperature of the condenser chamber(cold trap) to around -50oC (it can only reach max of -53oC). I then load samples in to the drying chamber that is then mounted on the mouth of the cold trap and switch on the vacuum pump. When the pump is put on the vacuum does not immediately begin, it delays for about a minute before it starts. And upon application of vacuum the samples are pushed up the vials(containers), melts , bubbles and pours off. What could be causing this?
This file probably helpful https://www.science.gov/topicpages/e/eutectic+freeze+crystallization.html
Dear Itseng,
I had a lot of trouble freeze drying samples in my PhD and there are three issues you may need to address.
First, it may be that your freeze dryer is not producing a strong enough vacuum and this, at least in the early stages of the freeze drying process, may allow samples to start to melt and bubble-up.
Second, it may take some time for the vacuum to be established, and during this period your samples may start to thaw because the air inside the vacuum bottles and the vacuum bottles themselves are at room temperature. You can fix this easily by placing the bottles in a freezer (-80°C if possible) to cool down.
Third, the movement of the sample up to the mouth of the tube is simply stopped if you freeze the sample at an angle; the more surface exposed to the vacuum, the faster the process will work. I used to make sure that all of my samples were in containers with lids (e.g. plastic tubes) and I put a small hole in each to allow gas movement. This prevented all of the sample from simply exploding out of the tube and into the vacuum bottle. In addition, try to have a big bottle compared to the sample tube size, as this will mean that the initial application of the vacuum will be less violent and less likely to move the sample.
I hope that this helps you,
Andrew
Very helpful observations Andrew J Spiers . The freeze dryer seem to produce enough vacuum but the challenge is it does not act immediately upon switching the pump on which leads to the second point. I have been chilling the drying chamber (vacuum bottle) in a cold room but you made me realize it is not enough it needs to be frozen. The kind of containers I am using may not be favourable for freezing at an angle although that can be very helpful. I need to freeze dry in glass vials which I seal under vacuum when the process ends because of the nature of the product and its use. I will however attempt to use long necked ampoules which can be frozen at an angle. Overall you have helped me fine tune some loose ends which will help me make some considerable progress
Much appreciation
Itseng
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