Any lyophilization experts here who might be able to help me with some problems?

Not an expret but with some expireance
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Well, I have been freeze-drying (I am freeze-drying a fungus) for a while now and I have tested some protectants.
The problem now is that some protectants do not seem to work and I need to be able to explain why they might fail.

The question now is why do the protectants not work.
The protectans are: glycerol (10%) , lactose (10%)+ skimmed milk (10%) , Fructose (10%).
When I used those substances the product just melts after freezing it. I freeze for about 30 minutes up to an hour in a methanol bath that is about -60°C and then I start drying it.
But instead of drying and getting the sublimation it just melts.

I was wondering why? Maybe because the temperature is too high when drying it?

I have other protectants that do work, like 10% sucrose with 1% gelatin and others...

How do you dry the sample?

What is the product you are trying to lyophilize?

I am freeze drying a fungus. Verticillium likely fungis.

How do I dry the samples? I dry them with a vacuum dryer.
I dont really know what you mean.
I use an old freeze dryer from virtis.(Freezemobile 5)
I simpely freeze the samples then I add them too the vacuum dryer. So its a vacuum thats dries the sample. I have no idea about the operation aspects (temperature of the vacuum systeem, or how low it goes in pressure) of the equipement I am using.

Here are a couple of resources that may help or provide useful information on the preservation of Fungi.

Long-Term Preservation of Fungus Cultures with
Liquid Nitrogen Refrigeration
SHUH-WEI HWANG
American Type Culture Collection, Rockville, Maryland
APPuE MICROBIOLOGY, Sept., 1966
Vol. 14, No. 5

http://aem.asm.org/cgi/reprint/14/5/784.pdf

Preservation and Recovery of Filamentous Fungi
ATCC
Link: http://www.atcc.org/common/documents/pdf/tb02.pdf

The sample melted because you didnt have enough vacuum. Check for leakage and do an oil change when needed. There should be a meter that tells you what kind of vacuum the system can deliver.

ghunter you are right, but also wrong.

It did indeed melt because there was a problem with the vacuum, but that is fixed now and new tests showed that it still melts, even with the fixed vacuum the samples with fructose, glucose and dmso melt.
The sample with the lactose and skimmed milk does work now.

So its still not fixed.
I think the temperature may also be an answer: I think the temperature need to stay lower to have a good sublimation.
But I cant keep the temperature any lower then it is now.

any other ideas?

What's the reading on vacuum? Did you rotate the flask to get a reasonably evenly distributed film of the sample when you froze it? or the room was too hot?

cant remember the vacuum reading, but that was not the problem anymore.

and yes, the flasks to rotate during the freezing.
Its evenly distributed.

I think its the temperature thas it too hot.
I do not cool the samples during sublimation, they are only cooled when I freeze them then I put them on the freezedrier and then they are not anymore cooled. They do heat up then during the sublimation just to roomtemperature.
I think I need to keep the samples cooled down to let the dmso, glycerol and fructose work.

On my hand, you need to get the value below 400 to keep it from melting. Try not to load too much sample at one time. Its the balancing act of the rate of water evaporation which brings heat out and keeps the sample frozen vs the room temp which melts your sample that determines your sample in the liquid or solid status. If you see a ice layer build-up on the outside of the flask, it is a good sign that you have a good vacuum going.

I do know that it is very important to keep the vacuum low, but the vacuum is working fine.

And I do not load a lot of sample at 1 time, only 10-20 ml.

The ice layer is almost always present at the outside when I freezedry, only when I use glycerol, dmso or fructose as a protectant it melts.
Other protectants work just fine.

So I think its something with the glycerol, dmso or fructose that is causing the freezing.
I think those products just need a lower temperature during the freezedrying to prevent them from melting.

I do know that glycerol and dmso especially are very good products to make the freezepoint very low, so I think its because of this that the samples melts: the freezingpoint is too low and the temperature of the sample is too high during the freezedrying after a while thus it melts.

I think what you experienced with glycerol and DMSO is understandable, coz they are liquid and they do lower the melting point, but for fructose....

yeah, indeed, dmso and glycerol is understandable, but fructose...

I do know that reducing sugars (like fructose) arent very good as a protectant, but that it would melt ? I find that strange.